A traditional Chinese medicine composition for treating cough variant asthma and application thereof

Abstract

The application provides a traditional Chinese medicine composition for treating cough variant asthma and application thereof, and is particularly applied to cough caused by wind evil invading the lung in traditional Chinese medicine. The composition comprises 6-15 parts of xuanfuhua, 6-15 parts of jingjie, 6-15 parts of qianhu, 10-20 parts of banxia, 6-15 parts of baishao, 3-9 parts of honeyed ma-huang and 3-9 parts of gancao, and has the effects of dispelling wind and relieving lung, reducing and stopping cough, resolving phlegm and relieving convulsion. The composition of the application has the synergistic effects of multiple components and multiple pathways, can reduce airway inflammation and remodeling of cough variant asthma and airway hyperresponsiveness, has fast effect, low recurrence rate and high safety. The composition can be prepared into a decoction, granules, capsules and the like, has simple preparation process, good stability and is suitable for clinical treatment and long-term conditioning of cough variant asthma.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a traditional Chinese medicine composition and its application for treating cough variant asthma. Background Technology

[0002] Cough variant asthma is a common chronic inflammatory lung disease, a special type of asthma. Its main clinical symptom is paroxysmal cough, often exacerbated at night or in the early morning, with particular sensitivity to hot, cold, and irritating stimuli and smoke. It may not be accompanied by wheezing or a normal bronchodilator test. Cough variant asthma has a high global incidence. A study as of 2020 showed that the prevalence of asthma in Chinese adults was approximately 1.81%, with cough variant asthma accounting for about 15.7% of all asthma patients. Furthermore, cough variant asthma accounts for approximately 25%–42% of chronic cough patients, making it a significant cause of chronic cough. In terms of treatment, the current principles are consistent with those for typical asthma, with inhaled corticosteroids combined with bronchodilators as the first-line treatment. While this can control acute symptoms to some extent, long-term use may induce systemic adverse reactions and carries a certain probability of progression to typical asthma, placing a heavy economic burden and medical pressure on patients and society. Therefore, exploring safe and effective treatment options is of great significance.

[0003] Traditional Chinese medicine (TCM) possesses advantages in multi-component, multi-target, multi-pathway, and holistic regulation, exhibiting stable clinical efficacy and few adverse reactions. The application of TCM in treating cough-variant asthma holds promising prospects and significant research value. According to TCM theory, cough-variant asthma falls under the category of "cough," with its core pathogenesis being impaired lung function and upward reversal of lung qi. Based on over ten years of clinical practice, the inventor has innovatively developed a TCM treatment for cough-variant asthma, particularly effective for cough-variant asthma caused by wind-evil invading the lungs and lung qi deficiency, incorporating fundamental TCM theories. Clinical and animal studies have demonstrated its excellent clinical efficacy.

[0004] The traditional Chinese medicine formula described in this invention consists of Inula japonica, Peucedanum praeruptorum, Schizonepeta tenuifolia, Pinellia ternata, Paeonia lactiflora, Ephedra sinica, and Glycyrrhiza uralensis. Its formulation closely addresses the pathogenesis of cough-variant asthma, working synergistically to dispel wind and clear the lungs, relieve cough, and alleviate spasms. This formula aligns perfectly with the principles of traditional Chinese medicine treatment for this disease and has shown excellent clinical efficacy in treating cough symptoms. Inula japonica is the principal herb, possessing a warm and salty nature, specifically entering the lung meridian. It also has the effects of resolving phlegm, lowering qi, and relieving cough, making it a key herb for treating cough caused by impaired lung function. Combined with Pinellia ternata, which dries dampness, resolves phlegm, lowers qi, and disperses stagnation, the two herbs together can remove phlegm and turbidity caused by lung qi stagnation, clear qi stagnation, and alleviate cough and phlegm symptoms. Schizonepeta tenuifolia, being pungent and warm, excels at dispelling wind-evil from the exterior, aiding in its expulsion. Peucedanum praeruptorum, with its descending and dispersing properties, works synergistically with Schizonepeta tenuifolia to restore the normal ascending and descending functions of lung qi. Ephedra sinica, with its pungent and warm properties, promotes lung function, relieves asthma and cough, effectively clearing lung qi. Combined with Inula japonica and Peucedanum praeruptorum, its ascending and descending functions are balanced, regulating qi and helping the lungs restore its normal functions of dispersing and descending. Furthermore, the combination of peony root and licorice root in the formula not only relieves spasms and alleviates the irritating dry cough caused by airway spasms, but also tonifies qi, harmonizes the middle jiao, protects the spleen and stomach, and prevents wind-dispelling drugs from depleting the body's vital energy. The entire formula is meticulously formulated, dispelling external wind to protect the lung's defensive qi and calming internal wind to eliminate phlegm and regulate qi, addressing both the root cause and the symptoms to achieve the effects of clearing the airways and restoring the normal ascending and descending functions of lung qi. This aligns with the TCM pathogenesis and clinical treatment needs of cough-variant asthma. Furthermore, clinical studies have shown no significant adverse reactions, making it a safe and effective treatment for cough variant asthma and a promising research direction. Summary of the Invention

[0005] 1. Purpose of the invention The purpose of this invention is to address the various drawbacks of Western medicine treatments mentioned above. Through research on traditional Chinese medicine, this invention provides a safe, effective, and long-term usable Chinese herbal composition and its applications. This Chinese herbal composition has the effects of dispelling wind and regulating qi, clearing the lungs and relieving cough, resolving phlegm and relieving spasms. It is used for cough-variant asthma caused by wind-evil invading the lungs, with symptoms such as: initial onset or prolonged cough, aggravated by wind, cold, or irritating odors; possibly accompanied by itchy throat, coughing up small amounts of thin white phlegm or no phlegm, nasal congestion, nasal itching, sneezing, etc.; pale tongue, white coating, and floating and tight or floating and wiry pulse. It has a rapid onset of action, low recurrence rate, and no obvious toxic side effects in treating cough-variant asthma. It can effectively improve symptoms, reduce airway hyperresponsiveness, and prevent progression to typical asthma.

[0006] To achieve the above objectives, the present invention provides the following solution.

[0007] 2. Composition of Traditional Chinese Medicine Composition

[0008] The traditional Chinese medicine composition of the present invention comprises the following raw materials in parts by weight: 6-15 parts of Inula japonica, 6-15 parts of Schizonepeta tenuifolia, 6-15 parts of Peucedanum praeruptorum, 10-20 parts of Pinellia ternata, 6-15 parts of Paeonia lactiflora, 3-9 parts of Ephedra sinica (processed with honey), and 3-9 parts of Glycyrrhiza uralensis.

[0009] The traditional Chinese medicine composition of the present invention is further optimized and contains the following raw materials in parts by weight: 10 parts of Inula japonica, 10 parts of Peucedanum praeruptorum, 10 parts of Schizonepeta tenuifolia, 15 parts of Pinellia ternata, 10 parts of Paeonia lactiflora, 6 parts of Ephedra sinica (honey-processed), and 6 parts of Glycyrrhiza uralensis.

[0010] 3. Pharmacological description of the raw material components in this invention

[0011] Inula japonica: This product has the effects of lowering qi and resolving phlegm, clearing the lungs and relieving cough, and is the principal drug; its chemical components mainly include cinnamic acid and its derivatives, flavonoids, flavanones such as cinnamyl glycoside, rutin, isorhamnetin, kaempferol, o-hydroxycinnamic acid and other components; isorhamnetin can reduce inflammatory factors such as IL-4, IL-5, and IL-13 in the airways of ovalbumin-induced asthma model mice, improve airway remodeling, and relieve lung inflammation in asthmatic mice; kaempferol can improve lung function in asthmatic mice and reduce inflammation and lung tissue damage.

[0012] Schizonepeta: This product has the effects of dispelling wind and releasing the exterior, and dispersing pathogenic factors. It is an assistant herb. Its chemical components mainly include cinnamic acid and its derivatives, flavonols, stigmasterol of flavonols, hesperidin, methyl rosmarinic acid, etc. Stigmasterol has the effect of reducing the attack symptoms of ovalbumin-induced asthma mouse model and improving its airway remodeling.

[0013] Peucedanum praeruptorum: This product has the effects of lowering qi and resolving phlegm, and dispersing wind-evil, and is used as an assistant herb; its chemical components mainly include simple coumarins, pyranocoumarins, ursane-type triterpenes and taraxane-type triterpenes such as peucedanin, peucedanin A, peucedanin C, furanocoumarins, etc.; studies have shown that peucedanin A has anti-inflammatory and antitussive effects, can reduce the symptoms of cough variant asthma model rats, regulate the secretion of inflammatory factors, and thus relieve cough variant asthma.

[0014] Pinellia ternata: This product has the effects of drying dampness and resolving phlegm, and dispersing stagnation and nodules. It is used as an adjuvant medicine. Its chemical components mainly include amino acids, purine nucleosides, and unsaturated fatty acids such as proline, methionine, oleic acid, and linoleic acid. Its fatty acids have anti-inflammatory, antioxidant, and immunomodulatory effects.

[0015] White peony root: This product has the effects of relieving spasms, expectorating phlegm and relieving cough, and is used as an adjuvant. Its chemical components mainly include monoterpenes, phenolic acids, flavonoids such as paeoniflorin, paeoniflorin lactone, benzoyl paeoniflorin, gallic catechin, etc. Paeoniflorin can regulate oxidative stress and autophagy mechanisms to alleviate inflammation and immune response in asthmatic mice, reduce the level of LPS-induced inflammatory factors in bronchial epithelial cells, and thus inhibit asthma inflammatory response.

[0016] Honey-processed ephedra: This product has the effects of pungent and warming the lungs, relieving asthma and cough, and is used as an adjuvant medicine. Its main chemical components include simple phenolic acids, phenylalanine-derived alkaloids such as norephedrine, ephedrine, protocatechuic acid, and p-hydroxybenzoic acid. Norephedrine can relax bronchial smooth muscle, reduce airway inflammation in asthma, and improve airway remodeling. Protocatechuic acid and other components can reduce inflammatory cell infiltration in mouse lung tissue and alleviate allergic asthma.

[0017] Licorice: This herb has the effects of tonifying the lungs and replenishing qi, moistening the lungs and relieving cough, and moderating the properties of other herbs. It is used as an adjuvant. Its main chemical components include glycyrrhizic acid, glycyrrhetinic acid, glycyrrhizin, and polysaccharides. Glycyrrhizin can downregulate the mRNA expression of IL-4 and IL-5 in mice through Th2-mediated cytokine signaling, exerting a potent anti-inflammatory and immunomodulatory effect on asthma. Glycyrrhizic acid can reduce airway inflammation and airway remodeling, alleviating chronic asthma in mice.

[0018] 4. Preparation method

[0019] Decoction: Weigh the raw materials according to the ratio, and package Inula japonica separately in a decoction bag; add 10 times the amount of water and soak for 30 minutes, bring to a boil over high heat, then simmer over low heat for 20-30 minutes, pour out the decoction, add 8 times the amount of water again and decoct in the same way, and combine the decoctions.

[0020] Ointment: The decoction is filtered, and the filtrate is concentrated under reduced pressure to 60°C, with a relative density of 1.10 to 1.20.

[0021] Other dosage forms: Extracts can be directly formulated into pharmaceutically acceptable dosage forms such as decoctions, mixtures, granules, capsules, and tablets.

[0022] Preferred process for granules: Add appropriate amounts of pharmaceutically acceptable excipients or auxiliary ingredients such as dextrin and stevioside to the extract, mix well, wet granulate, dry at 60-70℃, granulate, and package.

[0023] 5. Beneficial effects

[0024] High degree of alignment with pathogenesis: It closely follows the core pathogenesis of cough variant asthma caused by latent wind, combining five methods of dispelling wind, clearing the lungs, lowering qi, relieving cough, and resolving phlegm, addressing both the symptoms and the root cause.

[0025] Highly effective in relieving cough and spasms: It quickly improves symptoms such as irritating dry cough, itchy throat, and severe cough at night. Animal experiments and preliminary clinical trials have shown that this invention can significantly reduce the frequency of coughing, reduce airway inflammation and remodeling, and improve lung function, which is superior to conventional Western medicine treatment.

[0026] Low recurrence rate: Eliminating pathogenic factors and strengthening the body's resistance reduces recurrent attacks and lowers the risk of progression to typical asthma.

[0027] Safe and reliable: The medicine has a mild taste and no obvious toxic side effects, making it suitable for long-term use by adults and children.

[0028] Various dosage forms: It can be made into ointments, capsules, tablets, granules, etc., which are convenient to take, have good stability, and high compliance. Attached Figure Description

[0029] Figure 1 Total ion chromatograms of traditional Chinese medicine compositions under positive and negative ion modes were obtained using UHPLC-Q-TOF / MS.

[0030] Figure 2 This provides a network pharmacology analysis of the intersection of drugs and diseases, related genes, and core targets.

[0031] Figure 3 This is a network diagram of drug-active ingredient-target for network pharmacology analysis.

[0032] Figure 4 This is a graph showing the enrichment of GO and KEGG in network pharmacology analysis.

[0033] Figure 5 This is a diagram showing the molecular docking results of network pharmacology analysis.

[0034] Figure 6 The graph shows the changes in rat body weight and the changes in the number of times rats cough.

[0035] Figure 7 This is a bar chart showing the serum inflammation and related cytokine expression levels.

[0036] Figure 8 Microscopic image of the pathological morphology of rat lung tissue.

[0037] Figure 9 Microscopic images of the expression of α-SMA, Collagen-1, and PI3K / AKT / mTOR / HIF-1α pathway-related proteins in lung tissue.

[0038] Figure 10 Electrophoresis diagram of protein expression related to the PI3K / AKT / mTOR / HIF-1α pathway in lung tissue. Detailed Implementation

[0039] The present invention will now be described in detail with respect to various exemplary embodiments and the terminology used. This detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, the numerical ranges in this invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as any smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] The experimental methods described in the following embodiments of the present invention, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of the present invention.

[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, and all such modifications and variations should fall within the protection scope defined by the claims of this invention. This specification and its embodiments are merely exemplary.

[0043] The following description is based on specific embodiments.

[0044] Example 1: Clinical efficacy verification of traditional Chinese medicine composition

[0045] 1.1 Research Subjects and Methods

[0046] 1.1.1 Case Source: The cases in this clinical trial were patients with wind-evil invading the lungs type of cough from the outpatient department of pulmonology from February 2025 to December 2025. They met the definition of cough in "Internal Medicine of Traditional Chinese Medicine (New Century Fourth Edition)"; according to the "Standards for Diagnosis and Efficacy of Diseases and Syndromes in Traditional Chinese Medicine", all of them had symptoms of wind-evil invading the lungs: initial or long-term cough, aggravated by wind, cold or smell of irritating odors, accompanied by itchy throat, no sputum or cough with a small amount of thin white sputum or nasal congestion, itchy nose, sneezing, pale tongue, white coating, floating and tight or floating and wiry pulse, etc.; all met the diagnostic criteria of cough variant asthma in the "Guidelines for Diagnosis and Treatment of Cough (2025)" formulated by the Asthma Group of the Respiratory Diseases Branch of the Chinese Medical Association: chronic cough, often accompanied by obvious nocturnal irritating cough, positive bronchodilator test or provocation test of pulmonary function test, or PEF average diurnal variability rate >10%; effective anti-asthma treatment, excluding other diseases that cause chronic cough.

[0047] 1.1.2 Grouping and treatment methods: In accordance with the principle of randomization and double-blindness, a random number table was generated using SPSS 25.0 software, and 100 patients were included and divided into an experimental group and a control group in a 1:1 ratio, with 50 patients in each group.

[0048] Experimental group: The drug granules of this invention (Inula japonica 10g, Paeonia lactiflora 10g, Peucedanum praeruptorum 10g, Pinellia ternata 15g, Schizonepeta tenuifolia 10g, Glycyrrhiza uralensis 6g, Ephedra sinica 6g) were administered after being brewed with boiling water, 3 times / day, for 2 months; at the same time, budesonide / formoterol powder inhaler (160ug / 4.5ug, provided by AstraZeneca Trading Co., Ltd.) was administered, 1 inhalation / time, 2 times / day.

[0049] Placebo control group: The drug granule form of the present invention was administered as a mimic (the ingredients are starch and other excipients plus 1 / 10 of the original formula, and the mimic is consistent with the drug in the experimental group in appearance, smell and taste), which was taken after being brewed with boiling water, 3 times / day, for 2 months; at the same time, budesonide formoterol powder inhaler (160ug / 4.5ug, provided by AstraZeneca Trading Co., Ltd.) was administered, 1 inhalation / time, 2 times / day.

[0050] 1.1.3 Evaluation of clinical efficacy and safety

[0051] The TCM syndrome scoring evaluation criteria were formulated with reference to the "Guiding Principles for Clinical Research of New Traditional Chinese Medicine Drugs" and were quantitatively assessed using a semi-quantitative scoring method. The core observation indicator, cough, was divided into three dimensions: cough frequency, cough severity, and cough nature, using a four-level scoring method (0 points for none, 3 points for mild, 6 points for moderate, and 9 points for severe). Secondary observation indicators included sputum production, itchy throat, shortness of breath, nasal congestion, nasal itching, and sneezing, with the same scoring method as cough, and the scoring standards being 0 points, 1 point, 2 points, and 3 points.

[0052] The efficacy evaluation combined the Simplified Cough Severity Scale (SCS), the Leicester Cough Scale (LCS) score, and lung function.

[0053] The overall TCM syndrome efficacy evaluation is based on a four-level classification standard: basically cured, significantly effective, effective, and ineffective. The total effective rate is the percentage of the sum of cases in the basically cured, significantly effective, and effective categories out of the total number of cases.

[0054] Safety indicators: symptoms and signs of the patient before and after medication; objective indicators: electrocardiogram, complete blood count, urinalysis, liver function, and kidney function.

[0055] All patients were assessed at least twice, before and after treatment.

[0056] 1.1.4 Statistical Methods: SPSS 25.0 software was used for statistical analysis. A p-value ≤ 0.05 was considered statistically significant. Quantitative data following a normal distribution were expressed as mean ± standard deviation. Independent samples were used for comparisons between groups. t The test, comparing pre- and post-treatment results within the same group, used paired samples. t Tests; for non-normal distributions, use quartiles and medians ( P 25, P 50, P75) indicates that inter-group comparisons were conducted using... Mann-WhitneyU Tests were conducted to compare pre- and post-treatment results within each group. WilcoxonZ Paired rank-sum test. For count data, expressed as frequency (ƒ) or relative frequency (%), use the chi-square test for comparison of four-fold table data; for ordinal data, use the rank-sum test for comparison.

[0057] 1.2 Research Results

[0058] 1.2.1 Baseline Data Comparison

[0059] There were no statistically significant differences in age, gender, and disease duration between the two groups of patients. P >0.05), which is comparable (see Table 1).

[0060]

[0061] 1.2.2 Comparison of therapeutic effect evaluation results

[0062] The overall clinical efficacy was compared after treatment. Among 93 patients, 47 were in the experimental group and 46 were in the control group. The total effective rate in the experimental group was 93.62%, which was higher than the total effective rate of 78.26% in the control group. P =0.023 < 0.05 (Table 2).

[0063]

[0064] Traditional Chinese Medicine Syndrome Scores: There was no statistically significant difference in scores between the two groups of patients before treatment. P =0.829>0.05), indicating comparability; both groups of patients experienced a decrease in scores after treatment ( P <0.001), the score reduction in the experimental group was more significant than that in the control group. P =0.015 < 0.05 (see Table 3)

[0065] Lung function: Comparison of FEV1 / FVC ratio: There was no statistically significant difference between the two groups of patients before treatment. P =0.354>0.05), indicating comparability; both groups showed elevated levels after treatment ( P <0.001), the increase in the experimental group was more significant than that in the control group ( P =0.018 < 0.05 (see Table 4).

[0066]

[0067] Comparison of peak expiratory flow (PEF): There was no statistically significant difference between the two groups of patients before treatment. P=0.750>0.05), indicating comparability; both groups showed elevated levels after treatment ( P <0.001), the increase in the experimental group was more significant than that in the control group ( P =0.041 < 0.05 (see Table 5).

[0068]

[0069] Comparison of simplified cough scores: There was no statistically significant difference between the two groups of patients before treatment. P =0.308>0.05), indicating comparability; both groups showed a decrease after treatment ( P <0.001), the experimental group showed a more significant decrease than the control group ( P =0.001 < 0.01 (see Table 6).

[0070]

[0071] Comparison of Leicester Cough Scale scores: There was no statistically significant difference between the two groups before treatment. P =0.308>0.05), indicating comparability; both groups showed an increase after treatment ( P <0.001), the experimental group showed a more significant increase than the control group ( P =0.001 < 0.01 (see Table 7).

[0072]

[0073] 1.2.3 Safety Indicators: In this study, vital signs such as body temperature, resting heart rate, respiration, and blood pressure were monitored in both groups before and after drug treatment, and no significant abnormalities were detected. Safety indicators, including complete blood count, liver function, kidney function, urinalysis, and electrocardiogram, were measured in both groups before and after drug treatment, and no significant abnormalities were observed. A total of 3 adverse events were recorded in this clinical trial, 1 in the experimental group and 2 in the control group. All were mild, and no serious adverse events were observed. All three cases involved oral leukoplakia, which was determined to be caused by failure to rinse the mouth after using budesonide / formoterol powder inhaler. Patients were instructed to rinse their mouths promptly after medication and to take antifungal medication for a short period, which improved their oral symptoms. All adverse events recorded in the clinical trial were mild and related to improper use of Symbicort, indicating that the drug has good safety.

[0074] Conclusion: The results of this clinical trial show that the combination of the invented traditional Chinese medicine composition and Western medicine can significantly alleviate patients' cough and related symptoms, improve lung function, and enhance their quality of life. The total effective rate reached 93.62%, significantly better than the 78.26% of the control group. P=0.023). Furthermore, the traditional Chinese medicine composition of the present invention exhibits good safety. Overall, it indicates that the traditional Chinese medicine composition of the present invention has the effects of dispelling wind and ventilating the lungs, lowering qi and relieving cough, and can safely and effectively treat cough variant asthma.

[0075] Example 2: Complete Chemical Composition Analysis of Traditional Chinese Medicine Composition

[0076] Three sets of repeated tests were performed on the same batch of traditional Chinese medicine composition with the same proportions as in Example 1, including the following steps:

[0077] The entire medicinal formula was decocted twice with 10 times and 8 times the amount of water, respectively. The decoctions were combined, filtered, and concentrated to 6.7g of dry extract. 3g of the dry extract was placed in a 50mL Erlenmeyer flask, 30mL of methanol was added, and the mixture was thoroughly vortexed and then ultrasonically emulsified for half an hour. The emulsion was filtered through a 0.22μm filter membrane, and the resulting clear liquid was placed in a sample vial for analysis.

[0078] Chromatographic conditions: The target compound was separated by chromatographic separation using a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. Phase A of the liquid chromatography column was isopropanol:acetonitrile (1:1); Phase B was aqueous phase containing 0.01% acetic acid. A gradient elution program was set as follows: 0–5 min, 5%–15% A; 5–10 min, 15%–35% A; 10–20 min, 35%–65% A; 20–30 min, 65%–95% A. The sample pan temperature was 4 °C, the flow rate was 0.2 mL / min, and the injection volume was 2 μL.

[0079] Mass spectrometry conditions: Electrospray ionization (ESI) was used, scanning in both positive and negative ion analysis modes. Sheath gas flow rate: 50 Arb, auxiliary gas flow rate: 15 Arb, ion transfer tube temperature: 320℃, full MS resolution: 60000, MS / MS resolution: 15000; collision energy gradients: 20, 30, 40 eV, spray voltage: 3.8 kV (positive), -3.4 kV (negative).

[0080] Three sets of sample solutions were scanned and analyzed in positive and negative ion modes. Based on the chromatographic retention time, quasi-molecular ion peaks, fragment ion information, and molecular formula of the compounds, combined with the BiotreeDB (V3.0) database, metabolite identification and visualization were performed using the R package XCMS (v4.1.12) and MetDNA (V2). A total of 355 chemical components were resolved and identified, including 196 shikimic acid and phenylpropionic acid derivatives, 63 terpenes, 29 fatty acids, 28 alkaloids, 21 amino acids and short peptides, 11 carbohydrates, 6 polyketides, and 1 trans-butenedioic acid. Positive and negative ion characteristic spectra are attached to the instruction manual. Figure 1 .

[0081] Conclusion: Using UHPLC-Q-TOF / MS technology, a variety of chemical components in the invented traditional Chinese medicine composition were systematically identified. The anti-inflammatory, antitussive, antioxidant, and immunomodulatory pharmacological effects of the main components, such as flavonoids, coumarins, ephedrine alkaloids, and paeoniflorin, are highly consistent with the core pathological processes of cough-variant asthma. The diversity and complexity of its active ingredients provide solid material evidence for the treatment of cough-variant asthma with the invented traditional Chinese medicine composition.

[0082] Example 3: Network pharmacological analysis of traditional Chinese medicine composition.

[0083] 3.1 Analytical Methods:

[0084] The active ingredients of seven traditional Chinese medicines in the herbal composition were retrieved using the TCMSP (Traditional Chinese Medicine Systems Pharmacology Database). Oral bioavailability ≥30% and drug-likeness ≥0.18 were used as thresholds for screening. Combined with the components of the herbal composition analyzed in Example 2, components with high pharmacokinetic and drug-likeness were screened using SwissADME. Drug targets were predicted using the SwissTargetPrediction database and TCMSP. Simultaneously, disease targets for cough-variant asthma were retrieved from the GeneCards, DisGeNET, and OMIM databases using "CoughVariantAsthma" as the keyword. The intersection of drugs and disease targets was imported into the STRING12.0 database to construct a protein-protein interaction network for screening key targets; a "drug-active ingredient-target" network was also constructed to screen key active ingredients. These key targets were then imported into the David database for GO functional annotation and KEGG enrichment analysis, with the highest significance (P<0.05) selected as the threshold. Finally, molecular docking validation was performed on the top five key components and core targets.

[0085] 3.2 Analysis Results:

[0086] 167 components meeting the criteria were screened from the TCMSP database. Combined with the components analyzed in Example 2, 221 effective components were screened out, and 1244 pharmacogenetic targets were predicted and screened. 930 highly correlated targets for cough-variant asthma were found; 265 targets showed an intersection between the two (see...). Figure 2 A, 2B). Protein-protein interaction network analysis identified 47 key genes (see...). Figure 2 C). The top 10 core targets by intensity include: TNF-α, IL6, IL1B, AKT1, SRC, EGFR, STAT3, NFKB1, CASP3, and BCL2 (see...). Figure 2D). Based on the "drug-active ingredient-target" network analysis, the top 5 active pharmaceutical ingredients are: 11α,12α-epoxy-3β-23-dihydroxy-30-norolean-20-en-28,12β-olide, MEGxp0_000407, Chryseriol, Cyclo(Ala-Val), and (-)-Catechin. These may be key targets for developing traditional Chinese medicine combinations to intervene in cough-variant asthma (see...). Figure 3 ).

[0087] Enrichment analysis yielded 550 GO entries and 151 KEGG pathways. GO biological processes mainly involve regulation of apoptosis, RNA polymerase II transcriptional regulation, inflammatory responses, and cell proliferation regulation; cellular components are mainly distributed in the cytoplasm, plasma membrane, nucleus, and extracellular regions; molecular functions are concentrated in protein binding, enzyme binding, and kinase activity; the top 10 most significant entries were visualized (see [link to relevant documentation]). Figure 4 A). KEGG enrichment focused on signaling pathways such as PI3K-AKT, HIF-1, AGE-RAGE, and mTOR, and the top 20 entries were visualized (see [link]). Figure 4 B).

[0088] Molecular docking results showed that, except for the docking of Il-6 with Cyclo (Ala-Val), the binding energies of all groups were less than -5 kcal / mol; more than half of the binding energies were ≤-7 kcal / mol, indicating that the binding ability of each key component to the core target was strong, suggesting that the invented traditional Chinese medicine combination can form a stable complex to exert a therapeutic effect on cough variant asthma (see...). Figure 5 ).

[0089] Conclusion: Network pharmacology analysis revealed that key active ingredients in the herbal composition of this invention, such as Chryseriol, 11alpha,12alpha-epoxy-3beta-23-dihydroxy-30-norolean-20-en-28,12beta-olide, MEGxp0-000407, Cyclo(Ala-Val), and (-)-Catechin, may exert a therapeutic effect on cough variant asthma by acting on the core targets of TNF-α, IL6, IL1B, AKT1, and SRC through signaling pathways such as PI3K-AKT, HIF-1, AGE-RAGE, and mTOR.

[0090] Example 4: In vivo animal verification of the efficacy and mechanism of the traditional Chinese medicine composition.

[0091] 4.1 Experimental Materials

[0092] Instruments: 402Ai ultrasonic nebulizer (Jiangsu Yuyue Medical Equipment Co., Ltd.), TGL16-WS high-speed refrigerated centrifuge (Hunan Xiangyi Technology Co., Ltd.), BX53 optical microscope (Olympus Corporation, Japan), Synergy HTX multi-functional microplate reader, 12-0625 electrophoresis apparatus (Bio-Rad Corporation, USA), QuickChemi 5200 chemiluminescence imaging system (Wuhan Mona Biotechnology Co., Ltd.).

[0093] Reagents: The same herbal composition as in Example 1 (Inula japonica 10g, Schizonepeta tenuifolia 10g, Peucedanum praeruptorum 10g, Pinellia ternata 15g, Paeonia lactiflora 10g, Glycyrrhiza uralensis 6g, Ephedra sinica 6g) was purchased from the Traditional Chinese Medicine Hospital of Beibei District, Chongqing. Prednisolone acetate tablets (Shandong Lukang Pharmaceutical Group Saite Co., Ltd., batch number H20023023). Ovalbumin and aluminum hydroxide (Shanghai Yuanye Biotechnology Co., Ltd., batch numbers: S12015-25g, S30353-500g, purity not less than 99%). Rat Immunoglobulin E (IgE), Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), Scr family tyrosine protein kinases (SrcPTKs), Epidermal growth factor receptor (EGFR), Malondialdehyde (MDA), Glutathione (GSH), and Superoxide dismutase (SOD) Enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog numbers ml003022, ml064292, ml002859, ml059385, ml003425, ml077384, ml531010, ml077379).Alpha-smooth muscle actin (α-smooth muscle actin, α-SMA) antibody (Wuhan Sanying Biotechnology Co., Ltd., catalog No. 14395-1-AP); Collagen type I (Collagen-I) antibody (Jiangsu Qinke Biological Research Center Co., Ltd., catalog No. AF7001); Hypoxia Inducible Factor-1 (HIF-1) alpha antibody (Wuhan Boster Biological Technology Co., Ltd., catalog No.: PB9253); Rabbit anti-Phosphoinositide 3-Kinase (PI3K), phosphorylated PI3K (Phosphorylated Phosphoinositide 3-Kinase, p-PI3K), Protein kinase B (AKT), phosphorylated AKT (Phosphorylated Protein Kinase B, p-AKT), mammalian Target Of Rapamycin (mTOR), phosphorylated mTOR (Phosphorylated mammalian Target of Rapamycin, p-mTOR), β-actin and Horseradish Peroxidase-labeled Goat Anti-Rabbit Immunoglobulin G antibody (Wuhan ABclonal Biotech Co., Ltd., with catalog numbers A4992, AP0427, A18120, AP1208, A2445, AP1413, AC038 and AS014, respectively).

[0094] Animals: Thirty-six SPF-grade female SD rats, aged 6 to 7 weeks, weighing (210±10) g, were purchased from Chongqing Ensiville Experimental Animal Sales Co., Ltd., with license No. SCXK (Xiang) 2021-0002. All rats were fed in an environment with an ambient temperature of 20-23°C, a relative humidity of 40%-60%, and 12-hour alternating light and dark cycles, with free access to food and water.

[0095] 4.2 Preparation of Traditional Chinese Medicine and Grouped Administration

[0096] Preparation of Traditional Chinese Medicine: Weigh the raw medicinal materials of the inventive traditional Chinese medicine composition according to the ratio, and package and encapsulate Inula japonica Thunb. in a separate decocting bag; add 10 times the amount of water to soak for 30 min, boil over strong fire then turn to low heat to decoct for 20-30 min, pour out the medicinal liquid, add 8 times the amount of water again and decoct by the same method, combine the decoctions, concentrate by suction filtration, to prepare water decoctions with mass concentrations of 3.5, 7, and 14 g / kg (calculated based on the total amount of prepared herbal pieces) for later use.

[0097] Grouping and Administration: Rats were randomly divided into 6 groups using a random number table: normal control group, model group, positive control group, and low, medium, and high dose traditional Chinese medicine (TCM) groups. Except for the normal control group, which received saline, each group received an intraperitoneal injection of 1 mL of a mixed solution of 10% ovalbumin and aluminum hydroxide on days 0 and 7 to induce sensitization. From day 14 onwards, 1% ovalbumin was administered via ultrasonic nebulization for 30 min / day for a total of 14 days. Treatment began on day 14. One hour before nebulization, the positive control group received prednisone acetate 0.9 mg / kg / day by gavage. The low, medium, and high dose TCM groups received 3.5 g / (kg·d), 7 g / (kg·d), and 14 g / (kg·d) of the drug solution, respectively (calculated as 0.5 times, 1 times, and 2 times the clinically equivalent dose of the TCM composition). The normal and model groups received an equal volume of saline.

[0098] 4.3 Experimental Analysis Methods

[0099] Body weight and cough frequency measurement: From day 0 to day 28 of modeling, rats were weighed every 2 days. 24 hours after the last administration, the rats were placed in a transparent box and nebulized with 0.1 mmol / L capsaicin solution for 1 min. After turning off the nebulizer, the rats were observed for another 4 min and the number of coughs within 5 min was recorded.

[0100] Serum inflammation and related cytokine expression: After anesthetizing rats with intraperitoneal injection of tribromoethanol (25 mL / kg), blood was collected via the abdominal aorta and serum was separated by centrifugation at 4°C. The levels of IgE, IL-6, TNF-α, SRC, EGFR, MDA, GSH, and SOD were detected according to the ELISA kit requirements.

[0101] Pathological observation of lung tissue: The right upper lobe of the lung of each group of rats was fixed with 4% paraformaldehyde, dehydrated, embedded and sectioned, and then stained with hematoxylin-eosin (HE), Masson and periodic acid-Schiff (PAS). The pathological changes of the bronchus and lung tissue were observed under an optical microscope.

[0102] Detection of α-SMA and Collagen-1 expression in lung tissue: Immunohistochemistry (IHC) was used for detection. Paraffin-embedded tissue sections were dewaxed, hydrated, and incubated overnight with the corresponding primary antibody. The next day, secondary antibody was added and cultured for 1 hour, followed by DAB staining. Tissue changes were observed under an optical microscope.

[0103] Detection of pathway-related protein expression in lung tissue: Combining network pharmacology analysis and previous literature reports, the PI3K / AKT / mTOR and HIF-1 signaling pathways were validated. The expression of related proteins was detected using IHC (as above) and Western blotting (WB). Right lower lung tissue from rats was lysed on ice to extract proteins. After quantification, the proteins were separated by electrophoresis, transferred to a membrane, and incubated overnight at 4°C with internal control and primary antibody. The next day, secondary antibody was added and incubated for 1 hour. Finally, ECL imaging analysis was performed.

[0104] Statistical analysis: SPSS 25.0 was used for statistical analysis. Normally distributed measurement data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. For post-hoc multiple comparisons, Tukey's test was used for homogeneous variances and Tamhane's T2 test was used for heterogeneous variances.

[0105] 4.4 Experimental Results

[0106] Body weight and cough frequency measurements: Compared with the normal group, the model group rats showed weight loss after day 14, followed by a slower rate of weight gain. Prednisolone acetate and different doses of traditional Chinese medicine showed a trend towards improved body weight. Compared with the normal group, the model group rats exhibited a significantly increased number of coughs. P <0.01); Compared with the model group, the number of coughs in each treatment group was significantly reduced ( P <0.01), and the efficacy of the traditional Chinese medicine group was dose-dependent (see Table 8). Figure 6 ).

[0107] Serum inflammation and related cytokine expression levels: Compared with the normal group, the levels of IgE, IL-6, TNF-α, SRC, EGFR, and MDA in the model group rats were significantly increased. P <0.01); Meanwhile, compared with the model group, except for IgE and MDA in the low-dose traditional Chinese medicine group, the above indicators were reduced in all treatment groups ( P <0.05 or P <0.01). Furthermore, GSH and SOD levels in the model group were significantly lower than in the normal group ( P <0.01); Except for the low and medium dose groups of traditional Chinese medicine, the levels of GSH and SOD in all treatment groups were significantly higher than those in the model group. P <0.01)(see Table 8) Figure 7 ).

[0108]

[0109] Pathological observation of lung tissue: HE staining results showed that the model group had extensive inflammatory cell infiltration around the airways and thickened airway epithelium; the airway inflammatory infiltration and epithelial changes were significantly reduced in all treatment groups, with the most significant improvement in the high-dose traditional Chinese medicine group (see...). Figure 8 A). Masson staining results showed a significant increase in peri-airway blue collagen deposition in the model group, suggesting airway fibrosis; collagen deposition was significantly reduced in all treatment groups (see...). Figure 8 B). PAS staining results showed increased airway epithelial mucin secretion and goblet cell proliferation in the model group, which were significantly inhibited after drug treatment (see [link to PAS staining results]). Figure 8 C).

[0110] Lung tissue α-SMA and Collagen-1 expression levels: Immunohistochemical analysis showed that the positive expression of α-SMA and Collagen-1 in the peri-airway region of rats in the model group was significantly enhanced. After treatment, their expression was lower than that in the model group, and the expression in the traditional Chinese medicine group was dose-dependent (see...). Figure 9 A, 9B).

[0111] Lung tissue pathway-related protein expression levels: Immunohistochemical analysis showed that the expression of phosphorylated p-PI3K, p-AKT, p-mTOR, and HIF-1α in the lung tissue of the model group rats was higher than that in the normal group, and decreased after treatment (see...). Figure 9 C~9F). Protein electrophoresis results showed that there were no significant differences in the total protein expression levels of PI3K, AKT, and mTOR in the lung tissues of rats from different groups; however, the protein expression levels of p-PI3K, p-AKT, p-mTOR, and HIF-1α, as well as the ratios of p-PI3K / PI3K, p-AKT / AKT, and p-mTOR / mTOR, were significantly increased in the model group rats. P <0.01); Compared with the model group, except for the low-dose traditional Chinese medicine group of p-AKT / AKT, the above indicators in all treatment groups were significantly reduced ( P <0.01), among which the traditional Chinese medicine groups showed a dose-dependent effect (see Table 9). Figure 10 ).

[0112]

[0113] Conclusion: The herbal composition of the present invention may improve cough variant asthma by regulating the PI3K / AKT / mTOR / HIF-1α signaling pathway, alleviating airway inflammation and remodeling, and regulating oxidative stress.

Claims

1. A traditional Chinese medicine composition for treating cough-variant asthma, characterized in that, The raw materials contain the following parts by weight: 6-15 parts of Inula japonica, 6-15 parts of Schizonepeta tenuifolia, 6-15 parts of Peucedanum praeruptorum, 10-20 parts of Pinellia ternata, 6-15 parts of Paeonia lactiflora, 3-9 parts of Ephedra sinica (processed with honey), and 3-9 parts of Glycyrrhiza uralensis.

2. The traditional Chinese medicine composition according to claim 1 is further optimized, characterized in that, The weight proportions of each raw material are as follows: 10 parts of Inula japonica, 10 parts of Peucedanum praeruptorum, 10 parts of Schizonepeta tenuifolia, 15 parts of Pinellia ternata, 10 parts of Paeonia lactiflora, 6 parts of Ephedra sinica (honey-processed), and 6 parts of Glycyrrhiza uralensis.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that... Includes the following steps: (1) Weigh the raw materials according to the proportions; add water and decoct 1-2 times, then combine the decoctions; (2) Filter the decoction; concentrate it to a specific density to prepare oral liquid and extract; (3) Dry the extract under vacuum at 60-70℃, pulverize, and sieve to obtain dry extract granules, which are then made into granules. (4) Further pulverize the dry extract granules, sieve them to obtain dry extract powder, and make capsules.

4. The preparation method according to claim 3, characterized in that, Each time, add water at a rate of 8 to 10 times the total weight of the medicinal materials, and decoct for 0.5 hours. Pass the decoction through a 200-mesh sieve, the dry extract granules through a 14-mesh sieve, and the dry extract powder through a 40-mesh sieve.

5. A pharmaceutical preparation for treating cough-variant asthma, characterized in that... It comprises the traditional Chinese medicine composition as described in claim 1 or 2 and pharmaceutically acceptable excipients.

6. The pharmaceutical preparation according to claim 5, characterized in that... The dosage forms of the preparation are decoction, granules, capsules, oral liquid, pills, and tablets.

7. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the treatment of cough variant asthma.

8. The application according to claim 7, characterized in that... The cough described is due to wind-evil invading the lungs.

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