Discovery method of cough-relieving and phlegm-eliminating quality marker of cough-relieving granules and quality marker

Abstract

The invention relates to the technical field of traditional Chinese medicine research, in particular to a discovery method of a cough relieving and phlegm eliminating quality marker of cough relieving granules and the quality marker. According to the application, the cough-relieving and phlegm-eliminating drug effects of the cough-relieving granules are evaluated by virtue of concentrated ammonia water cough inducing and phenol red excretion tests, plasma pharmaceutical chemistry research of the cough-relieving granules is developed by virtue of a UHPLC-Q-Exactive Plus Orbitrap HRMS technology, and 26 prototype blood-entering components of the cough-relieving granules are analyzed and identified; through network pharmacological analysis of components entering blood, 11 potential active components for relieving cough and eliminating phlegm of the cough-relieving granules are screened, relate to 40 common targets such as IL6, TLR4, STAT3 and the like, and mainly participate in signal channels such as PI3K-Akt, HIF-1, EGFR (epidermal growth factor receptor) and the like; the seven prototype blood-entering components are quantitatively analyzed by utilizing pharmacokinetics. The three compounds of azelaic acid, caffeic acid and vanillin are used as the cough relieving and phlegm eliminating quality markers of the cough relieving granules through effectiveness, transitivity and testability. The research result is of great significance in deeply defining the pharmacodynamic material basis of the cough-relieving granules, optimizing the quality standard and promoting the clinical application of the cough-relieving granules.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine research, and in particular to a method for discovering a quality marker of Kening granules for relieving cough and removing phlegm, and a quality marker. Background Art

[0002] Kening Granule is the main Chinese medicine variety of Guizhou Dalong Pharmaceutical Co., Ltd. It is a pure Chinese medicine OTC preparation composed of three medicinal materials: pine cone, loquat leaf, and cotton root. It does not contain central antitussive drugs. It has the effects of relieving cough and expectoration, relieving asthma, and strengthening the body. It is included in the national drug standard [WS3-B-1581-93]. It is a classic prescription for "using both cold and hot". It has been clinically used for cough, cold, chest tightness, cold, cough, chronic bronchitis and other symptoms for many years, with significant efficacy. As early as in the folk, there was a Dong medicine "Ganoderma lucidum pine cone cotton root soup", which has the effects of invigorating qi and nourishing blood, relieving cough and relieving asthma. It is used to treat chronic bronchitis, and the clinical efficacy is as high as 96.7%. Pine cone decoction is used to treat chronic bronchitis, and it is found that it has the effects of tonifying the kidney and strengthening yang, strengthening the body and eliminating evil, eliminating cold and dampness in the lungs, and improving the body's immune function. Cotton root has good expectorant, antitussive and qi-invigorating effects, and loquat leaf has the effects of clearing the lungs and reducing phlegm, relieving cough and asthma, and harmonizing the stomach and relieving adverse reactions, and has a long history of treating cough. The above studies show that Kening Granules have good market prospects and development potential in treating cough and sputum.

[0003] In November 2020, the "Technical Guidelines for Research on Quality Standards for New Chinese Medicines (Trial)" issued by the Center for Drug Evaluation of the State Drug Administration emphasized that quality standards should be based on medicinal materials as an important research content, focusing on the safety and effectiveness of drugs, and combining the transfer and traceability of the quantity and quality of Chinese medicine, so as to control the quality of medicinal materials as a whole and ensure the stability of preparation quality. The guidelines also pointed out that the selection of quality control methods for Chinese medicine should be tailored to the medicine, encourage the integration of multiple methods, and establish multiple indicators of inspection and testing items in the quality standards of Chinese medicine compound preparations. At present, the material basis of the efficacy of Kening Granules is unclear, and the current quality standards only have thin layer identification of two medicinal materials, pine cone and cotton root. There is a lack of effective quality control standards, and it is difficult to ensure the stability of preparation quality.

[0004] Since Academician Liu Changxiao proposed the new concept of quality markers (Q-Marker) for traditional Chinese medicine, the research methods of quality markers have been expounded from five aspects: effectiveness, specificity, transfer and traceability, measurability, and compatibility environment. They have been widely used in the quality control research of traditional Chinese medicines and their preparations. Based on the theoretical premise that the absorption of drug components into the blood is the drug effect, the plasma pharmacological chemistry research method is used to systematically analyze the transition components between the drug-preparation-blood to clarify the "transfer and traceability" of quality; the network pharmacology research method is used to construct the "traditional Chinese medicine-blood component-disease-target" network based on the blood-entering components, and the chemical components related to the disease are found, so that candidate quality markers with "effectiveness" can be screened; only the components that enter the body and can reach an effective concentration are more likely to be key quality markers. Through pharmacokinetic research, the dynamic changes of the absorption degree, concentration change, and residence time of the efficacy-related components in the body can be clarified. It has a certain guiding significance to use pharmacokinetic markers with "measurability" as candidate quality markers. Based on this, and on the basis of the chemical composition analysis and attribution of Kening Granules conducted by the research team in the early stage, this study will carry out ammonia-induced cough and phenol red excretion experiments to verify its cough and expectorant effects, and determine the quality markers of Kening Granules for cough and expectorant through plasma medicinal chemistry, network pharmacology and pharmacokinetic results, to provide a basis for in-depth exploration of the pharmacological material basis and quality control of Kening Granules. Summary of the invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for discovering a quality marker of the antitussive and expectorant of Kening granules and a quality marker, including the following contents:

[0006] A method for discovering a quality marker of relieving cough and removing phlegm in Kening granules comprises the following steps:

[0007] (1) Analyze and identify the prototype blood-entering components of Kening Granules;

[0008] (2) Using the prototype blood-entering components as the “drug component set”, the potential active components and common targets of Kening Granules for relieving cough and expectoration were screened through network pharmacology methods;

[0009] (3) Based on the analysis of blood-entering components, the in vivo process of the prototype blood-entering components is quantitatively analyzed using pharmacokinetics;

[0010] (4) The quality markers of the cough and expectorant effects of Kening Granules were finally determined through effectiveness, transferability, and measurability.

[0011] Furthermore, the Kening granules are composed of three medicinal materials: pine cone, loquat leaf, and cotton root. Kening granules are the main Chinese medicine varieties of Guizhou Dalong Pharmaceutical Co., Ltd. It is a pure Chinese medicine OTC preparation composed of three medicinal materials: pine cone, loquat leaf, and cotton root, and does not contain central antitussive drugs. It has the effects of relieving cough and expectoration, relieving asthma, and strengthening the body, and is included in the national drug standard [WS3-B-1581-93].

[0012] Furthermore, the step (1) of analyzing and identifying the prototype blood-entering components of the Kening granules is performed by using a 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 ° C, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2: 1) solution (B), injection volume: 2 μ L, 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), the spray voltage was 3500 V, and the ion lens voltage frequency was 50.0; the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, the nebulization temperature was 350°C, the capillary temperature was 320°C, the probe heater temperature was 350°C, and the scanning mode was full scan + data-dependent secondary scan (full scan + ddMS2) positive and negative ion exchange mode, the scanning range was m / z 80-1200, the primary resolution was 70000, and the secondary resolution was 17500.

[0017] Furthermore, in the UHPLC-Q-Exactive Plus Orbitrap HRMS method, the plasma to be tested is treated by the following method: 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 protein, vortexed for 5 min, ultrasonicated for 10 min, centrifuged at 8000 r / min at 4°C for 10 min in a refrigerated high-speed centrifuge, the supernatant is placed in a centrifuge tube, and blown dry with a nitrogen blower at 37°C to obtain the residue, 200 μL of 50% methanol is added to re-dissolve, vortexed for 2 min, ultrasonicated for 10 min, centrifuged at 12000 r / min at 4°C for 10 min, and the supernatant is collected as the sample solution for analysis by the UHPLC-Q-Exactive Plus Orbitrap HRMS instrument.

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

[0019] ① Screening of potential active ingredients and collection of targets of Kening Granules:

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

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

[0022] By entering the keywords "Cough" and "Sputum" in the GeneCards database for retrieval, disease-related targets were obtained, and the duplicate genes and genes without Uniprot ID were eliminated to obtain targets related to cough and expectoration;

[0023] ③Protein interaction network analysis and core target screening:

[0024] The Venny online software was used to intersect the potential active ingredient targets of the drugs with the disease targets and draw a Venn diagram, so as to obtain the common targets of the potential active ingredients for treating cough and expectoration. The intersection targets were imported into the String database to obtain the interaction relationship between the target proteins. The PPI network diagram was drawn and the topological structure analysis was performed using the Analyze network tool and the CytoNCA plug-in in the Cytoscape3.10.0 software to obtain the degree value (Dgree), betweenness centrality (Betweenness), and closeness centrality (Closeness) topological parameters of each node and calculate the top 20 targets respectively. The intersection targets obtained by the three algorithms are the core targets;

[0025] ④ Construction and analysis of the “drug-ingredient-disease-shared target” network:

[0026] The data of potential active ingredients, diseases and common target genes of drugs were processed to generate Network and type files, which were then imported into Cytoscape 3.10.0 software to draw a "drug-ingredient-disease-common target" network diagram;

[0027] ⑤Enrichment analysis:

[0028] The intersection targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) and enrichment analysis was performed on the Gene Ontology database "Gene ontology, abbr. GO" and the Kyoto Encyclopedia of Genes and Genomes "Kyoto Encyclopedia of Genes and Genomes, abbr. KEGG". Then, bubble plots and rectangular plots were created through the online drawing platform Weishengxin, and biological interpretations were performed.

[0029] Furthermore, in step (3), the in vivo process of the prototype entering the blood component is quantitatively analyzed by pharmacokinetics, and the specific conditions are as follows:

[0030] Chromatographic conditions:

[0031] Column: ACQUITY BEH C18 (2.1×50 mm, 1.7 μm); guard column: Waters Van Guard BEH C18 (2.1×5 mm, 1.7 μm); mobile phase: A: 0.2% formic acid in water, B: 0.2% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 1 μL; gradient elution conditions are as follows:

[0032] Table 1 Mobile phase gradient elution table

[0033]

[0034] Mass spectrometry conditions:

[0035] Electrospray ion source (ESI), 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 monitoring ions of each component are as follows:

[0036] Table 2 Multiple reaction ion monitoring (MRM) mass spectrometry conditions

[0037]

[0038] Furthermore, in the step (3), the sample to be tested is prepared by the following method: 200 μL of plasma is taken and placed in a 1.5 mL plastic centrifuge tube, 40 μL of 40 ng / mL puerarin (internal standard) solution, 20 μL of 1% formic acid water and 800 μL of acetonitrile are added in sequence, vortexed for 3 min, ultrasonicated for 10 min, centrifuged at 12000 r / min for 10 min at 4 ° C, the supernatant is separated and dried with nitrogen at 37 ° C, the residue is dissolved with 200 μL of 50% acetonitrile aqueous solution, vortexed for 3 min, ultrasonicated for 10 min, centrifuged at 14000 r / min for 10 min at 4 ° C, and the supernatant is analyzed on an ACQUITY UPLC I-Class / Xevo TQ-S ultra high performance liquid phase tandem triple quadrupole coupling instrument.

[0039] A quality marker for relieving cough and removing phlegm in Kening granules, which 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 method to analyze and identify the following components in plasma: nicotinamide, DL-norleucine, adenine, adenosine, 2,5-dihydroxybenzoic acid, maltol, 8-hydroxyquinoline, homovanillic acid, caffeic acid, riboflavin, vanillin, 4-hydroxycinnamic acid, benzodihydrofuran-2-carboxylic acid, scopoletin, 4-indolecarboxaldehyde, azelaic acid, 3-tert-butyladipic acid, andrographolide, cafestol, 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, cafestol, 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 ° C, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2: 1) solution (B), injection volume: 2 μ L, 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), the spray voltage was 3500 V, and the ion lens voltage frequency was 50.0; the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, the nebulization temperature was 350°C, the capillary temperature was 320°C, the probe heater temperature was 350°C, and the scanning mode was full scan + data-dependent secondary scan (full scan + ddMS2) positive and negative ion exchange mode, the scanning range was m / z 80-1200, the primary resolution was 70000, and the secondary resolution was 17500.

[0046] A method for detecting multiple components in plasma, and simultaneously determining the contents 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 (2.1×50 mm, 1.7 μm); guard column: Waters Van Guard BEH C18 (2.1×5 mm, 1.7 μm); mobile phase: A: 0.2% formic acid in water, B: 0.2% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 1 μL; gradient elution conditions are as follows:

[0050] Table 1 Mobile phase gradient elution table

[0051]

[0052] Mass spectrometry conditions:

[0053] Electrospray ion source (ESI), 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 monitoring ions of 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 created by the present invention are embodied in:

[0057] (1) This application evaluates the antitussive and expectorant effects of Kening Granules through concentrated ammonia-induced cough and phenol red excretion tests, and uses UHPLC-Q-Exactive Plus Orbitrap HRMS method to analyze and identify 26 prototype blood-entering components of Kening Granules.

[0058] (2) This application uses network pharmacology methods to screen out 11 potential active ingredients of Kening Granules for relieving cough and removing phlegm. They exert their cough and expectorant effects mainly by acting on 40 common targets involving IL6, TLR4, STAT3, etc., and are involved in the regulation of signaling pathways such as PI3K-Akt, HIF-1, and EGFR.

[0059] (3) This application uses pharmacokinetics to quantitatively analyze the in vivo processes of 7 prototype blood-entering components. Through effectiveness, transmissibility, and measurability, three compounds, azelaic acid, caffeic acid, and vanillin, were used as quality markers for the cough and expectorant effects of Kening Granules. The results of this study are of great significance for further clarifying the material basis of the efficacy of Kening Granules, optimizing quality standards, and promoting its clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is the effect of Kening granules on cough in mice. Note: A: Comparison of cough latency in mice of each group, B: Comparison of cough times within 5 minutes in mice of each group, M: model group; DEX: dextromethorphan hydrobromide group; KNKL-L, KNKL-M, KNKL-H are low, medium, and high groups of Kening granules, respectively. Compared with the model group, # P<0.05, ## P<0.01, ### P<0.001.

[0061] Figure 2is the tracheal phenol red secretion of mice in each group. Note: NC: normal control group; AMB: ambroxol hydrochloride group; KNKL-L, KNKL-M, KNKL-H are the low, medium and high groups of Kening granules, respectively. Compared with the control group, *P<0.05, **P<0.01.

[0062] Figure 3 This is the chromatogram of drug-containing plasma and blank plasma of Kening Granules. Note: A. Blank plasma-ESI+; B. Blank plasma-ESI-; C. Drug-containing plasma-ESI+; D. Drug-containing plasma-ESI-.

[0063] Figure 4 It is a Venn diagram of the genes that intersect the active ingredients of Kening Granules with cough and sputum.

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

[0065] Figure 6 It is a visualization diagram of the active ingredients of Kening Granules and the intersection targets of cough.

[0066] Figure 7 This is the network diagram of "drug-ingredient-common target-disease" of Kening Granules.

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

[0068] Fig. 9 It is a rectangular diagram of GO biological function enrichment analysis.

[0069] Fig.10 It is a KEGG pathway enrichment bubble map.

[0070] Fig.11 UPLC-MS / MS chart of each component.

[0071] Fig.12 This is the average concentration-time curve of seven compounds in rat plasma after oral administration of Kening granules. DETAILED DESCRIPTION

[0072] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0073] Example:

[0074] Study on the quality markers of Kening granules for relieving cough and expectoration based on plasma pharmacology combined with 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 a production license number of SCXK(Xiang)2022-0011; SD rats (250±20g) were purchased from Changsha Tianqin Biotechnology Co., Ltd. with a production license number of SCXK(Xiang)2019-0014. Both were SPF-grade animals and were kept in an environment with a light / dark cycle, 20-24°C, and a relative humidity of 50-70% for one week before being used in experimental studies. They had free access to water and food during the period. All experimental studies were approved by the Animal Ethics Committee of Guizhou Medical University.

[0078] 1.2 Reagents and instruments

[0079] Phenol red (Tianjin Komiou 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 (Sinopharm Chemical Reagent Co., Ltd., batch number: 20230329); dextromethorphan hydrobromide oral solution (Zhuhai Federal Pharmaceutical Co., Ltd. Zhongshan Branch, batch number: 31044111); heparin sodium (Beijing Solebao Biotechnology Co., Ltd., batch number: NO.1031R021); puerarin (China Food and Drug Inspection Institute, batch number: 110752-202217); methanol, formic acid, acetonitrile (Merck, Germany, chromatographic grade); α-linolenic acid, azelaic acid, caffeic acid (Sichuan Weikeqi 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 Biotechnology 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 phase tandem triple quadrupole coupling instrument including binary gradient pump, autosampler, column oven, triple quadrupole mass analyzer and Masslynx 4.1 mass spectrometry workstation (Waters, USA); EL204 1 / 10,000 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.); KQ500DE numerically controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); THZ-82 water bath constant temperature oscillator; Allegra X-30R low temperature high speed centrifuge (Beckman Coulter, USA); Model 680 microplate reader (Bio-Rad Life Sciences Products Co., Ltd.); YLS-8A multifunctional cough and wheeze inducing instrument (Jinan Yiyan Technology Development Co., Ltd.); nitrogen blowdown instrument (Beijing Zhongxing Huili Technology Development Co., Ltd.).

[0081] 1.3 Database 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 cough experiment in mice

[0085] 2.1.1 Modeling and drug administration

[0086] Thirty-six mice, half male and half female, were randomly divided into 6 groups: normal control group (NC), model group (M), dextromethorphan hydrobromide group (clinical equivalent dose 18.2 mL / kg, DEX) and low (clinical 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 Kening granules, with 6 mice in each group. The mice were fasted for 12 hours before administration. The normal control group and model group were given the same volume of normal saline, and the other groups were gavaged at 20 mL / (kg·bw), twice a day, for 7 consecutive days. One hour after the last administration, the mice were placed in the YLS-8A cough and wheeze induction instrument, and the normal control group was sprayed with 0.9% normal saline by atomization, and the other groups were sprayed with 28% ammonia water by atomization (replace 5 mL of fresh ammonia water before each animal is placed) for 45 seconds.

[0087] 2.1.2 Detection indicators

[0088] The cough latency (s) and the number of coughs within 5 min were recorded (based on the mouse's abdominal muscles contracting violently, shoulders and back shrugging with mouth opening, and coughing sounds), and the cough suppression rate was calculated.

[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 5 groups: normal control group (NC), ambroxol hydrochloride group (163.8 mg / kg, AMB), and low (clinical 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 Kening Granules, with 6 mice in each group. The blank control group was given an equal volume of normal saline, and the mice in the other groups were gavaged twice a day for 7 consecutive days. 30 minutes after the last administration, 5% phenol red solution was injected intraperitoneally at a rate of 10 mL / (kg·bw). 30 minutes later, the mice were killed by dislocating the neck and fixed on the operating board in the supine position. The skin of the neck 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 normal saline containing 0.1 mL of 1 mol / L sodium hydroxide (NaOH), shaken, ultrasonicated for 5 minutes, shaken for 15 minutes, and centrifuged at 4000 r / min at 4°C for 5 minutes. The supernatant was transferred to a centrifuge tube for later use.

[0092] 2.2.2 Detection indicators

[0093] Take 200 μL of plasma supernatant in a 96-well plate and measure the OD value at 546 nm on an enzyme-labeled instrument.

[0094] The phenol red content was calculated by regression equation. Drawing of standard phenol red curve: 0.01 g of phenol red was accurately weighed on an analytical balance, dissolved in 5% sodium bicarbonate (NaHCO3), and prepared into 100 μg / mL phenol red solution, and then gradiently diluted into 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 10 μg / mL phenol red solution, and the absorbance was measured at 546 nm with an enzyme marker, and the phenol red concentration was used as the horizontal axis and the OD value as the vertical axis to draw a standard curve, and the excretion of phenol red was calculated according to the regression equation.

[0095] 2.3 Statistical analysis

[0096] Measurement data are presented as mean ± standard deviation SPSS23 statistical software was used for one-way ANOVA analysis. If the variance was homogeneous, LSD multiple comparison was used for inter-group comparison; if the variance was unequal, Dunnett's T3 multiple comparison was used for inter-group comparison. P < 0.05 was considered statistically significant.

[0097] 2.4 Analysis of blood components of Kening granules

[0098] 2.4.1 Chromatographic conditions

[0099] Chromatographic column: CORTECS UPLC T3 (2.1 mm×100 mm, 1.6 μm), column temperature: 40°C, 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), the spray voltage was 3500 V, and the ion lens voltage frequency was 50.0; the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, the nebulization temperature was 350°C, the capillary temperature was 320°C, the probe heater temperature was 350°C, and the scanning mode was full scan + data-dependent secondary scan (full scan + ddMS2) positive and negative ion exchange mode, the scanning range was m / z 80-1200, the primary resolution was 70000, and the secondary resolution was 17500.

[0102] 2.4.3 Collection and processing of biological samples

[0103] 14 mice were randomly divided into a blank control group (2 mice) and a Kening granule administration group (clinical equivalent dose 91g / kg) of 12 mice. The Kening granule administration group was divided into administration 15min, 30min, 45min, 60min, 90min, 120min groups to analyze the blood components at multiple time points, 2 mice in each group. Fasting for 12h before the experiment, free drinking water, Kening granules were administered by gavage according to the weight of mice, twice a day, for 7 consecutive days, and the blank control group was gavaged with normal saline. After the last administration, blood was collected from the eyeballs at 15, 30, 45, 60, 90, 120min after administration, respectively, and placed in a 1.5mL centrifuge tube coated with 1% sodium heparin, centrifuged at 4000r / min, 4℃ for 10min, and the supernatant was taken to obtain blank plasma and drug-containing plasma. The drug-containing plasma at each time point was mixed and stored at -20℃ for standby use.

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

[0105] 2.5 Network pharmacology analysis

[0106] 2.5.1 Screening of Potential Active Ingredients and Collection of Targets of Kening Granules

[0107] The components entering the blood were imported into the SwissADME database to screen for potential active ingredients, and then the Swiss TargetPrediction database was used to limit the species to "Homo sapiens" to predict the targets of potential active ingredients and delete invalid duplicate targets.

[0108] 2.5.2 Collection of cough and expectorant related targets

[0109] Disease-related targets were obtained by entering the keywords "Cough" and "Sputum" respectively into the GeneCards database for retrieval, and then the targets related to cough and expectoration were obtained by eliminating duplicate genes and genes without Uniprot ID.

[0110] 2.5.3 Protein interaction network analysis and core target screening

[0111] The Venny online software was used to intersect the potential active ingredient targets of the drugs with the disease targets and draw a Venn diagram, so as to obtain the common targets of the potential active ingredients for treating cough and expectoration. The intersection targets were imported into the String database to obtain the interaction relationship between the target proteins. The PPI network diagram was drawn and the topological structure analysis was performed using the Analyze network tool and the CytoNCA plug-in in the Cytoscape3.10.0 software to obtain the degree value (Dgree), betweenness centrality (Betweenness), and closeness centrality (Closeness) topological parameters of each node and calculate the top 20 targets respectively. The intersection targets obtained by the three algorithms are the core targets.

[0112] 2.5.4 Construction and analysis of the “drug-ingredient-disease-shared target” network

[0113] The data of potential active ingredients of drugs, diseases and common target genes were processed to generate Network and type files, which were then imported into Cytoscape 3.10.0 software to draw the "drug-ingredient-disease-common target" network diagram.

[0114] 2.5.5 Enrichment analysis

[0115] The intersection targets were imported into the DAVID database (https: / / david.ncifcrf.gov / ) and enrichment analysis was performed on the Gene Ontology database "Gene ontology, abbr. GO" and the Kyoto Encyclopedia of Genes and Genomes "Kyoto Encyclopedia of Genes and Genomes, abbr. KEGG". Then, bubble plots and rectangular plots were created through the online drawing platform Weishengxin, 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, add methanol to dissolve and dilute to the scale to obtain a 1 mg / mL puerarin stock solution, accurately pipette an appropriate amount of the stock solution and dilute it to a 40 ng / mL puerarin internal standard solution, and store it at -20 °C for later use.

[0119] Weigh the appropriate amount of nicotinamide, 2,5-dihydroxybenzoic acid, α-linolenic acid, azelaic acid, caffeic acid, vanillin, and riboflavin reference substances accurately, add methanol to dissolve them, and prepare a single reference substance stock solution with a mass concentration of 1 mg / mL. Accurately pipette appropriate amounts of the above-mentioned single reference substance solutions of different volumes, dilute and fix the volume with 50% methanol, and prepare a mixed reference substance solution containing 7 components with mass concentrations of nicotinamide 1μg / mL, 2,5-dihydroxybenzoic acid 0.3μg / mL, α-linolenic acid 0.3μg / mL, azelaic acid 0.1μg / mL, caffeic acid 0.3μg / mL, vanillin 1μg / mL, and riboflavin 1μg / mL, and store at -20℃ for later use.

[0120] 2.6.2 Sample collection and processing

[0121] Eight healthy male SD rats were fasted for 12 h before administration and were allowed to drink water freely. They were given Kening granules (at a dose of 91 g / kg) by single oral gavage. About 0.5 ml of blood was collected from the fundus venous plexus at 0.083, 0.167, 0.25, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, and 36 h after administration. The blood was placed in a heparin-coated centrifuge tube and centrifuged at 4°C, 12000 r / min for 10 min. The supernatant was taken and stored in a -80°C refrigerator for later use.

[0122] 200 μL of rat plasma was taken and placed in a 1.5 mL plastic centrifuge tube, and 40 μL of 40 ng / mL puerarin (internal standard) solution, 20 μL of 1% formic acid water and 800 μL of acetonitrile were added in sequence, and the mixture was vortexed for 3 min, ultrasonicated for 10 min, and centrifuged at 12000 r / min for 10 min at 4 °C. The supernatant was separated and dried with nitrogen at 37 °C, and the residue was dissolved with 200 μL of 50% acetonitrile aqueous solution, vortexed for 3 min, ultrasonicated for 10 min, and centrifuged at 14000 r / min for 10 min at 4 °C. The supernatant was analyzed on an ACQUITY UPLC I-Class / Xevo TQ-S ultra-high performance liquid phase tandem triple quadrupole coupling instrument.

[0123] 2.6.3 Chromatographic conditions

[0124] Column: ACQUITY BEH C 18 (2.1×50 mm, 1.7 μm); Guard column: Waters Van GuardBEH C 18 (2.1×5 mm, 1.7 μm); mobile phase: A: 0.2% formic acid in water, B: 0.2% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 1 μL. Gradient elution conditions are shown in Table 1.

[0125] Table 1 Mobile phase gradient elution table

[0126]

[0127]

[0128] 2.6.4 Mass spectrometry conditions

[0129] Electrospray ion source (ESI), capillary voltage 3 kV, ion source temperature 120 ° C, desolvation gas (nitrogen) 1000 L / H, desolvation temperature 600 ° C, collision gas (argon) 0.15 mL / min; multiple reaction ion monitoring (MRM) mode for positive and negative ion synchronous monitoring, each component monitoring ion is shown in Table 2.

[0130] Table 2 Multiple reaction ion monitoring (MRM) mass spectrometry conditions

[0131]

[0132] 2.6.5 Methodological investigation

[0133] Specificity investigation: The chromatograms of blank plasma, blank plasma plus 7 mixed reference substances and IS solution, and plasma of oral Kening granules plus IS solution were analyzed to evaluate the specificity.

[0134] Linear range and lower limit of quantitation: Accurately draw the reference substance stock solution, mix it, and dilute it step by step with methanol to prepare the mixed reference substance solution. Take 200 μL of blank blood, add 10 μL of each gradient mixed reference substance, process it according to the plasma sample method, and evaluate the linear relationship.

[0135] Precision and Accuracy Four concentrations of quality control samples (n=5) were tested on the same day and on three consecutive days to examine the intra-day and inter-day precision and accuracy of the method.

[0136] Matrix effect and extraction recovery rate investigation The peak area of ​​the quality control sample added to the blank plasma before and after treatment was compared to investigate the extraction recovery rate. The mixed reference substance in plasma was compared with the mixed reference substance in pure solvent to investigate the matrix effect.

[0137] The stability study evaluated the changes in the chemical properties of the quality control samples in plasma under different storage conditions, including short-term stability at 4°C for 12 hours, freeze-thaw stability after 3 freeze-thaw cycles, and stability at -20°C for 24 hours.

[0138] 2.6.6 Statistical data processing

[0139] The pharmacokinetic parameters of each component were fitted using the non-compartmental analysis method in WinNonLin 8.2.2 (Phoenix, Pharsight, USA) data processing software.

[0140] 3 Results

[0141] 3.1 Effects of Kening Granules on Cough Latency and Cough Frequency in Mice Induced by Ammonia

[0142] After ammonia stimulation, mice showed obvious coughing reaction. Compared with the model group, the cough latency of the positive drug group and the low, medium and high dose groups of Kening Granules was significantly prolonged (P<0.001), and the number of coughs within 5 minutes was significantly reduced (P<0.05, P<0.01, P<0.001). From the cough suppression rate, the cough suppression effect of Kening Granules did not show obvious dose dependence. The results suggest that Kening Granules have a good inhibitory effect on ammonia-induced cough, as shown in Table 3 and Figure 1 .

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

[0144]

[0145] 3.2 Effect of Kening Granules on Tracheal Phenol Red Excretion in Mice

[0146] In this experiment, the phenol red standard curve is y = 0.0768x + 0.0388, R 2 =0.9999. The phenol red excretion of each group was calculated by regression equation. The results showed that compared with the control group, the phenol red excretion of the low-dose and medium-dose Kening granules groups increased (P < 0.05), and the ambroxol hydrochloride group and the high-dose Kening granules group increased compared with the control group, but the difference was not statistically significant, as shown in Table 4 and Figure 2 shown.

[0147] Table 4 Effect of Kening granules on phenol red secretion in mouse trachea (mean ± SD, n = 6)

[0148]

[0149]

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

[0151] 3.3 Analysis of blood components of Kening granules

[0152] Based on the qualitative analysis of the chemical composition of Kening granules, FreeStyle TM1.3 software was used to analyze the data, and the chromatographic peaks in the samples were extracted through pre-processing such as peak extraction, peak matching, and background subtraction. By comparing the mass spectrometric characteristics of the mice in the drug-treated group, the blank control group, and the extract of Kening Granules, 26 prototype blood-entering components were detected in the drug-containing plasma of Kening Granules. The results are shown in Table 5. The chromatogram is shown in Figure 3 shown.

[0153] Table 5 Chromatographic peaks of the plasma prototype of Kening Granules entering the blood

[0154]

[0155]

[0156]

[0157]

[0158] 3.4 Network pharmacology analysis of blood-entering components

[0159] 3.4.1 Obtaining the intersectional targets of the potential active ingredients of Kening Granules and their antitussive and expectorant-related targets

[0160] The blood components were imported into the Swiss ADME database for screening, and 13 potential active ingredients were obtained. Probability>0 was used as the standard for Swiss Target Prediction to include targets. After deduplication, a total of 479 related targets were obtained. Disease-related targets were retrieved through the GeneCards database, and 2336 cough targets and 790 sputum targets with "Relevance Scores" greater than the median were screened out. After deduplication and gene standardization, 1897 cough targets and 698 sputum targets were obtained. A total of 40 targets were obtained using Venny2.1.0 online software, and a Venn diagram was drawn, see Figure 4 .

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

[0162] The 40 common targets were imported into the String database, the protein species was "Homo sapiens", the interaction score was limited to >0.4, and the single target (CPA3) without interaction was removed. The other parameters were kept at the default values ​​to obtain protein interactions. Figure 5 The protein interaction visualization diagram was constructed using Cytoscape 3.10.0 software. Figure 6The core targets were obtained by Dgree, Betweenness, and Closeness algorithms, and it was speculated that the following 17 targets were mainly affected by Kening Granules in the treatment of cough and expectoration: IL6, TLR4, STAT3, PTGS2, EGFR, ERBB2, MMP9, PTPRC, MAPK1, MTOR, BRAF, ACE, MPO, CCND1, MMP2, PIK3CG, and NR3C1.

[0163] 3.4.3 “Drug-ingredient-disease-shared target” network construction and obtaining effective substance groups

[0164] like Figure 7 As shown in the figure, the "drug-ingredient-disease-shared target" network diagram of Kening Granules for treating cough and expectoration is displayed. There are 11 ingredients in Kening Granules, including 8-Hydroxyquinoline, Homovanillic acid, Vanillin, 2,3-Dihydro-1-benzofuran-2-carboxylic acid, 4-Coumaric acid, Scopoletin, Caffeic acid, Azelaic acid, Andrographolide, Kahweol, and (+ / -)-Gingerol, which are involved in the regulation of cough and expectoration. The remaining two ingredients, Nicotinamide and Adenine, are not involved in the regulation. These 11 ingredients constitute the effective substance group of Kening Granules for treating cough and expectoration.

[0165] 3.4.5 Discovery of the antitussive and expectorant pathway of Kening Granules

[0166] The intersection targets were imported into the DAVID database for GO and KEGG enrichment analysis to explain the biological process (Biological Process, abbr. BP), cellular component (Cellular Component, abbr. CC) and molecular function (Molecular Function, abbr. MF) involved in the targets ( Figure 8 , 9 ) and obtained the main involved signaling pathways ( Fig.10). Results: 224 BP entries for cough and expectorant drugs were obtained, which were mainly related to positive regulation of protein modification and phosphorylation, metabolic process of phosphorus and phosphate, hormone regulation, etc.; 28 CC entries were obtained, mainly involving lysosomes, nuclear membrane, endoplasmic reticulum lumen, cell basement membrane, etc.; 36 MF entries were obtained, involving protein kinase binding and activity, phosphotransferase activity, etc.; mainly involving the following signaling pathways: PI3K-Akt signaling pathway, HIF-1 signaling pathway, coronavirus disease-COVID-19, Kaposi's sarcoma-associated herpes virus infection, endocrine resistance, EGFR tyrosine kinase inhibitor resistance, relaxin signaling pathway, ErbB signaling pathway, etc.

[0167] 3.5 Pharmacokinetic analysis of the components of Kening granules entering the blood

[0168] 3.5.1 Exclusivity

[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, and the components were well separated and had good peak shapes, indicating that the method had good specificity. Fig.11 .

[0170] 3.5.2 Standard curve, linear range and sensitivity

[0171] The seven components including nicotinamide in rat plasma had good linear relationship within their linear range, and the correlation coefficient (R2) of the standard curve of each component was greater than 0.99, as shown in Table 6.

[0172] Table 6 Linear regression curve equations of 7 ingredients including niacinamide

[0173]

[0174] 3.5.3 Accuracy and precision

[0175] The intra-day and inter-day precision RSDs of nicotinamide and other seven components in plasma were 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 nicotinamide and other seven components in plasma samples at three concentrations were in the range of 91.15% to 102.16%, and the matrix effects were in the range of 99.49% to 109.74%. The results showed that the extraction recoveries were good and there was no obvious matrix effect, which met the requirements of the biological sample analysis method.

[0178] 3.5.5 Stability

[0179] The RE values ​​of the seven components in the concentration samples after the plasma samples were stored at -20°C for 24 hours, placed at 4°C for 12 hours, and subjected to three freeze-thaw cycles (room temperature of -20°C) were in the range of -15 to 15%, and the RSDs were all less than 15%, indicating that the plasma biological samples containing these seven components had good stability after being stored at -20°C for 24 hours, placed at 4°C for 12 hours, and subjected to three freeze-thaw cycles.

[0180] 3.5.6 Pharmacokinetic parameters

[0181] The above established UPLC-MS / MS analysis method was used to determine the average blood drug concentration-time curve of normal rats after oral administration of Kening granules at various time points. Fig.12 , WinNonLin 8.2 software was used to calculate the pharmacokinetic parameters, and the non-compartmental model was fitted for the kinetic process of the drug in rats. The relevant pharmacokinetic parameters are shown in Table 8. After oral administration of Kening granules to normal rats, nicotinamide, riboflavin, caffeic acid, azelaic acid and other components reached peak concentrations before 3 hours, and were absorbed quickly. The half-life T1 / 2 of riboflavin and caffeic acid was less than 3 hours, indicating that the two had a short in vivo action time. The half-life T1 / 2 of nicotinamide and azelaic acid was greater than 10 hours, indicating that the two had a long in vivo action time; vanillin was rapidly absorbed in the body and had a short action time; 2,5-dihydroxybenzoic acid and α-linolenic acid reached peak concentrations around 12 hours, and the half-life T1 / 2 was greater than 10 hours, indicating that the two were slowly absorbed in the body and had a long action time.

[0182] Table 7 Pharmacokinetic parameters of the seven components after oral administration of Kening Granules

[0183]

[0184]

[0185] 4 Discussion

[0186] The quality control of Chinese herbal compound preparations is an important prerequisite for ensuring clinical efficacy. Therefore, this study is of great significance to explore the quality markers of antitussive and expectorant of Kening Granules based on in vivo efficacy, plasma drug chemistry combined with network pharmacology and pharmacokinetics. The antitussive and expectorant effects of Kening Granules were verified for the first time through the classic model of ammonia-induced cough and phenol red excretion experiments, confirming the effectiveness of Kening Granules. Using UHPLC-Q-Exactive Plus Orbitrap HRMS technology, 26 blood-entering components of the prototype of Kening Granules were analyzed, and the substances that entered the blood circulation and may be effective were determined, reflecting the traceability and transferability of the material basis of the efficacy. The network pharmacology was used to screen 13 potential active ingredients for the prototype blood-entering components, of which 11 components, including 8-hydroxyquinoline, homovanillic acid, vanillin, benzodihydrofuran-2-carboxylic acid, p-hydroxycinnamic acid, scopoletin, caffeic acid, azelaic acid, andrographolide, cafestol, and 6-gingerol, participated in the regulation and constituted the effective substance group for the treatment of cough and expectoration in Kening Granules. Through the "component-target-pathway" visualization network analysis, it was found that the main targets involved were IL-6, STAT3, EGFR, and other targets, and KEGG was enriched to PI3K-Akt, HIF-1 and other signaling pathways. It is suggested that the cough and expectoration effect of Kening Granules is through the joint action of multiple components and multiple targets. Based on the analysis of blood-entering components, the pharmacokinetic quantitative detection of the changes in the body of 7 components, including azelaic acid, nicotinamide, vanillin, caffeic acid, 2,5-dihydroxybenzoic acid, α-linolenic acid and riboflavin, reflects the measurability of the substance. Studies have found that caffeic acid has anti-influenza virus effects and is a potential active substance in the herb Huanyang ginseng for antitussive, antiasthmatic, anti-inflammatory and hypoglycemic effects. Azelaic acid has good anti-inflammatory, antioxidant, antimicrobial and anti-hemolytic activities. Fang Wei found azelaic acid in the blood migration components of the Huatanjiangqi formula into rats with chronic obstructive pulmonary disease model, which may be a potential active substance for the formula to reduce phlegm and reduce qi. Vanillin has anti-inflammatory and antitussive effects, plays a pre-protective role in acute lung injury, and reduces the protein expression levels of IL-6 and TNF-α inflammatory factors by regulating ERK, p38, AKT and NF-κB, thereby reducing inflammation. The above studies have confirmed the effectiveness of caffeic acid, azelaic acid and vanillin.

[0187] In summary, the results of this study showed that Kening Granules had significant antitussive and expectorant effects, and based on the definition of quality markers, caffeic acid, azelaic acid and vanillin were used as quality markers for antitussive and expectorant of Kening Granules through effectiveness, transmissibility and measurability. This result is of great significance for in-depth clarification of the pharmacological material basis of Kening Granules, optimization of quality standards and promotion of its clinical application.

[0188] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A method for discovering a quality marker for relieving cough and removing phlegm in Kening granules, characterized in that: The steps include: (1) Analyze and identify the prototype blood-entering components of Kening Granules; (2) Using the prototype blood-entering components as the "drug component set", the potential active components and common targets of Kening Granules for relieving cough and expectoration were screened through network pharmacology methods; (3) Based on the analysis of blood-entering components, the in vivo process of the prototype blood-entering components is quantitatively analyzed using pharmacokinetics; (4) The quality markers of the cough and expectorant effects of Kening Granules were finally determined through effectiveness, transferability, and measurability.

2. The method for discovering the quality marker of the Kening granules for relieving cough and eliminating phlegm according to claim 1, characterized in that: The Kening granules are composed of three medicinal materials: pine cones, loquat leaves, and cotton roots.

3. The method for discovering the quality marker of the Kening granules for relieving cough and removing phlegm according to claim 1, characterized in that: The step (1) analyzes and identifies the prototype blood-entering components of Kening Granules by using UHPLC-Q-Exactive Plus Orbitrap 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 ° C, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2: 1) solution (B), injection volume: 2 μ L, 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 was electrospray ionization (ESI), the spray voltage was 3500 V, and the ion lens voltage frequency was 50.0; the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, the nebulization temperature was 350°C, the capillary temperature was 320°C, the probe heater temperature was 350°C, and the scanning mode was full scan + data-dependent secondary scan (full scan + ddMS2) positive and negative ion exchange mode, the scanning range was m / z 80-1200, the primary resolution was 70000, and the secondary resolution was 17500.

4. The method for discovering the quality marker of the Kening granules for relieving cough and removing phlegm according to claim 3, characterized in that: In the UHPLC-Q-Exactive Plus Orbitrap HRMS method, the plasma to be tested is treated by the following method: 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 protein, vortexed for 5 min, ultrasonicated for 10 min, centrifuged at 8000 r / min at 4°C for 10 min in a refrigerated high-speed centrifuge, the supernatant is placed in a centrifuge tube, blown dry with a nitrogen blower at 37°C to obtain a residue, 200 μL of 50% methanol is added to re-dissolve, vortexed for 2 min, ultrasonicated for 10 min, centrifuged at 12000 r / min at 4°C for 10 min, and the supernatant is collected as a sample solution for analysis by a UHPLC-Q-Exactive Plus Orbitrap HRMS instrument.

5. The method for discovering the quality marker of the Kening granules for relieving cough and removing phlegm according to claim 1, characterized in that: The step (2) specifically comprises the following steps: ① Screening of potential active ingredients and collection of targets of Kening Granules: The blood-entering components were imported into the SwissADME database to screen for potential active ingredients, and then the SwissTargetPrediction database was used to limit the species to "Homo sapiens" to predict the targets of potential active ingredients and delete invalid and duplicate targets; ②Collection of targets related to cough relief and expectoration: By entering the keywords "Cough" and "Sputum" in the GeneCards database for searching, disease-related targets were obtained, and the genes with duplicates and no Uniprot ID were eliminated to obtain targets related to cough and expectoration. ③Protein interaction network analysis and core target screening: Use Venny online software to intersect the potential active ingredient targets of the drugs with the disease targets and draw a Venn diagram to obtain the common targets of the potential active ingredients for treating cough and expectoration. Import the intersection targets into the String database to obtain the interaction relationship between the target proteins. Use the Analyze network tool and CytoNCA plug-in in Cytoscape3.10.0 software to draw the PPI network diagram and perform topological structure analysis, obtain the degree value, betweenness centrality, and closeness centrality topological parameters of each node, and calculate the top 20 targets respectively. The intersection targets obtained by the three algorithms are the core targets; ④ "Drug-ingredient-disease-shared target" network construction and analysis: The data of potential active ingredients, diseases and common target genes of drugs were processed to generate Network and type files, which were then imported into Cytoscape 3.10.0 software to draw a "drug-ingredient-disease-common target" network diagram; ⑤Enrichment analysis: The intersection targets were imported into the DAVID database, and enrichment analysis was performed on the Gene Ontology database "Gene ontology, abbr. GO" and the Kyoto Encyclopedia of Genes and Genomes "Kyoto Encyclopedia of Genes and Genomes, abbr. KEGG". Then, bubble charts and rectangular charts were created through the online drawing platform Weishengxin, and biological interpretation was performed.

6. The method for discovering the quality marker of the Kening granules for relieving cough and removing phlegm according to claim 1, characterized in that: The step (3) uses pharmacokinetics to quantitatively analyze the in vivo process of the prototype blood-entering component, and the specific conditions are as follows: Chromatographic conditions: Chromatographic column: BEH C18 (2.1×50 mm, 1.7 μm); guard column: Waters VanGuard BEH C18 (2.1×5 mm, 1.7 μm); mobile phase: A: 0.2% formic acid in water, B: 0.2% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 1 μL; gradient elution conditions are as follows: Table 1 Mobile phase gradient elution table Mass spectrometry conditions: Electrospray ion source, capillary voltage 3kV, ion source temperature 120℃, desolvation gas 1000L / H, desolvation gas temperature 600℃, collision gas 0.15mL / min; multiple reaction ion monitoring mode for positive and negative ion synchronous monitoring, the monitoring ions of each component are as follows: Table 2 Multiple reaction ion monitoring (MRM) mass spectrometry conditions 7. The method for discovering the quality marker of the antitussive and expectorant of Kening granules according to claim 1, characterized in that: In the step (3), the sample to be tested is prepared by the following method: 200 μL of plasma is taken and placed in a 1.5 mL plastic centrifuge tube, 40 μL of 40 ng / mL puerarin (internal standard) solution, 20 μL of 1% formic acid water and 800 μL of acetonitrile are added in sequence, vortexed for 3 min, ultrasonicated for 10 min, centrifuged at 12000 r / min for 10 min at 4°C, the supernatant is separated and dried with nitrogen at 37°C, the residue is dissolved with 200 μL of 50% acetonitrile aqueous solution, vortexed for 3 min, ultrasonicated for 10 min, centrifuged at 14000 r / min for 10 min at 4°C, and the supernatant is analyzed on an ACQUITY UPLC I-Class / Xevo TQ-S ultra-high performance liquid phase tandem triple quadrupole coupling instrument.

8. A quality marker for relieving cough and removing phlegm of Kening granules, characterized in that: It is one or more of caffeic acid, azelaic acid and vanillin.

9. A method for detecting multiple components in plasma, characterized in that: The following components in plasma were analyzed and identified by UHPLC-Q-Exactive PlusOrbitrap HRMS method: nicotinamide, DL-norleucine, adenine, adenosine, 2,5-dihydroxybenzoic acid, maltol, 8-hydroxyquinoline, homovanillic acid, caffeic acid, riboflavin, vanillin, 4-hydroxycinnamic acid, benzodihydrofuran-2-carboxylic acid, scopoletin, 4-indolecarboxaldehyde, azelaic acid, 3-tert-butyladipic acid, andrographolide, cafestol, 6-gingerol, 2-Hydroxy-4,5',8a'-trimethyl-1'-oxo-4-vinyloctahydro-1'H-spiro[cyclopen tane-1,2'-naphthalene]-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-octahydrophena nthrene-1-carboxylic acid, cafestol, 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 ° C, flow rate: 0.3 mL / min, mobile phase: 0.1% formic acid aqueous solution (A) - acetonitrile methanol (2: 1) solution (B), injection volume: 2 μ L, 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 was electrospray ionization (ESI), the spray voltage was 3500 V, and the ion lens voltage frequency was 50.0; the sheath gas pressure was 35 arb, the auxiliary gas pressure was 10 arb, the nebulization temperature was 350°C, the capillary temperature was 320°C, the probe heater temperature was 350°C, and the scanning mode was full scan + data-dependent secondary scan (full scan + ddMS2) positive and negative ion exchange mode, the scanning range was m / z 80-1200, the primary resolution was 70000, and the secondary resolution was 17500.

10. A method for detecting multiple components in plasma, characterized in that: The levels of azelaic acid, nicotinamide, vanillin, caffeic acid, 2,5-dihydroxybenzoic acid, α-linolenic acid, and riboflavin in plasma were also determined; The specific conditions are as follows: Chromatographic conditions: Chromatographic column: BEH C18 (2.1×50 mm, 1.7 μm); guard column: Waters VanGuard BEH C18 (2.1×5 mm, 1.7 μm); mobile phase: A: 0.2% formic acid in water, B: 0.2% formic acid in acetonitrile; flow rate: 0.3 mL / min; column temperature: 40°C; injection volume: 1 μL; gradient elution conditions are as follows: Table 1 Mobile phase gradient elution table Mass spectrometry conditions: Electrospray ion source, capillary voltage 3kV, ion source temperature 120℃, desolvation gas 1000L / H, desolvation gas temperature 600℃, collision gas 0.15mL / min; multiple reaction ion monitoring mode for positive and negative ion synchronous monitoring, the monitoring ions of each component are as follows: Table 2 Multiple reaction ion monitoring (MRM) mass spectrometry conditions

Images