Application of genipin-1-beta-D gentiobioside in preparation of medicine for treating chronic obstructive pulmonary disease

Abstract

The invention relates to an application of genipin-1-beta-D gentiobioside in preparation of a medicine for treating chronic obstructive pulmonary disease. The genipin-1-beta-D gentiobioside is prepared into an aerosol inhalation solution, and the aerosol inhalation administration mode is adopted, so that the lung function in the chronic obstructive pulmonary disease can be improved, the content of inflammatory factors in lung tissues can be reduced, and the lung tissue injury can be relieved.

Description

Technical Field

[0001] This invention relates to the application of genipin-1-β-D gentiopicroside, and more particularly to the application of genipin-1-β-D gentiopicroside in the preparation of a drug for treating chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a common chronic airway disease characterized by persistent cough, sputum production, dyspnea, and shortness of breath. The pathogenesis of COPD includes: 1. Airway inflammation: Multiple cells participate in airway inflammation in COPD, including neutrophils, lymphocytes, and eosinophils. Neutrophils are the main effector cells. 2. Oxidative stress: Cigarette smoke and other inhaled particles generate oxygen free radicals. Simultaneously, COPD patients experience a decrease in endogenous antioxidant production, thereby activating inflammatory genes, inactivating antiproteases, stimulating mucus hypersecretion, and reducing the anti-inflammatory activity of glucocorticoids. 3. Protease / antiprotease imbalance: Elastin is a major component of lung parenchymal connective tissue. Proteases cause elastin destruction, a significant and irreversible cause of emphysema. 4. Emphysema: Overinflation and expansion of the lungs reduce lung tissue elasticity, often leading to the formation of bullae, causing changes in respiratory function. Impaired ventilation and gas exchange can result in hypoxia and carbon dioxide retention, leading to respiratory failure. Chronic obstructive pulmonary disease (COPD) can be divided into acute exacerbations and stable phases based on its disease progression.

[0003] Treatment for COPD includes medication, non-pharmacological therapies (such as pulmonary rehabilitation, smoking cessation, and oxygen therapy), and, in some cases, surgical intervention. Medications primarily include bronchodilators, inhaled corticosteroids, and anticholinergic drugs, designed to relieve airway spasms and reduce inflammation.

[0004] However, bronchodilators used in drug treatment may cause side effects such as palpitations, arrhythmia, and muscle spasms; inhaled corticosteroids may cause oral and pharyngeal candidiasis, and long-term use may increase the risk of osteoporosis, cataracts, and glaucoma; anticholinergic drugs may cause side effects such as dry mouth, urinary retention, and constipation.

[0005] Genipin-1-β-D-gentioside is a naturally occurring compound belonging to the iridoid class of compounds. It is one of the main active ingredients in gardenia (Gardenia jasminoides Ellis) and possesses various biological activities, including anti-inflammatory, hepatoprotective, choleretic, and hypoglycemic effects. Existing Chinese patent CN102000102A discloses the application of genipin-1-β-D-gentioside in the preparation of drugs for treating heart failure, and Chinese patent CN104510747B discloses the application of genipin-1-β-D-gentioside in the preparation of drugs with protective efficacy against pneumonia caused by influenza virus FM1 strain. However, research on the application of genipin-1-β-D-gentioside in the treatment of chronic obstructive pulmonary disease is not disclosed. Summary of the Invention

[0006] The purpose of this invention is to provide the application of genipin-1-β-D gentiopicroside in the preparation of a drug for treating chronic obstructive pulmonary disease (COPD). The genipin-1-β-D gentiopicroside of this invention has the characteristics of improving lung function in COPD, reducing the content of inflammatory factors in lung tissue, and alleviating lung tissue damage.

[0007] The technical solution of the present invention: the application of genipin-1-β-D gentiopicroside in the preparation of drugs for treating chronic obstructive pulmonary disease.

[0008] In the aforementioned application of genipin-1-β-D gentiopicroside, the genipin-1-β-D gentiopicroside is administered via nebulized inhalation.

[0009] In the aforementioned application of genipin-1-β-D gentiopicroside, the concentration of the nebulized inhalation solution of genipin-1-β-D gentiopicroside is 75 mg / ml or 37.5 mg / ml, the nebulization flow rate is 7.5 L / min, and the nebulization time is 0–30 min.

[0010] In the aforementioned application of genipin-1-β-D gentiopicroside, the nebulized inhalation concentration of 75 mg / ml genipin-1-β-D gentiopicroside is 2.419 mg / L.

[0011] In the aforementioned application of genipin-1-β-D gentiopicroside, the nebulized inhalation concentration of genipin-1-β-D gentiopicroside at 37.5 mg / ml is 1.032 mg / L.

[0012] In the aforementioned application of genipin-1-β-D gentiopicroside, the nebulized inhalation solution of genipin-1-β-D gentiopicroside comprises the following raw materials: genipin-1-β-D gentiopicroside, pH adjuster, osmotic pressure adjuster, and solvent.

[0013] In the aforementioned application of genipin-1-β-D gentiopicroside, the method for preparing the nebulized inhalation solution of genipin-1-β-D gentiopicroside includes the following steps:

[0014] (1) Measure out 40% to 90% of the total volume of the solvent required to prepare the solution to obtain the first solution;

[0015] (2) Add an osmotic pressure regulator to the first solution and stir until homogeneous to obtain the second solution;

[0016] (3) Add a pH adjuster to the second solution to adjust the pH value to 4.5-7.0 to obtain the third solution;

[0017] (4) Add genipin-1-β-D gentiopicroside to the third solution, stir well, and add a pH adjuster to keep the pH value between 4.5 and 7.0 to obtain the fourth solution;

[0018] (5) Add solvent to the fourth solution and bring the volume up to the required total volume of the solution to be prepared. Stir well to obtain the nebulized inhalation solution of genipin-1-β-D gentiopicroside.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The genipin-1-β-D gentiopicroside nebulized inhalation solution used in this invention has two concentrations, 75 mg / ml and 37.5 mg / ml. Within 0 min to 30 min of nebulization, the median aerosol particle size is between 2 and 3 μm, which meets the requirement that the particle size of the pulmonary administration inhalation solution is <3 μm.

[0021] During the nebulized inhalation concentration test, within 5 to 35 minutes of nebulization, the filter membrane concentration of 75 mg / ml genipin-1-β-D gentioside remained stable at 594.133–615.776 μg / ml, and the filter membrane concentration of 37.5 mg / ml genipin-1-β-D gentioside remained stable at 238.814–271.001 μg / ml, indicating that the content of genipin-1-β-D gentioside remained stable during nebulization, with nebulized inhalation concentrations of 2.419 mg / L and 1.032 mg / L, respectively. After nebulization for 20 minutes at the two concentrations of 75 mg / ml and 37.5 mg / ml genipin-1-β-D gentioside, the actual doses administered to rats were 37.53 mg / kg and 16.01 mg / kg, respectively.

[0022] This invention uses rat models of chronic obstructive pulmonary disease (COPD), rat models of acute exacerbation of COPD (AECOPD), ammonia-induced cough model in mice, and a mouse airway phenol red excretion and expectoration model to evaluate the efficacy of genipin-1-β-D gentiopicroside nebulized inhalation solution at two concentrations (75 mg / ml and 37.5 mg / ml) in the treatment of chronic obstructive pulmonary disease.

[0023] In a rat model of chronic obstructive pulmonary disease (COPD), genipin-1-β-D gentiopicroside, administered via nebulized inhalation for 20 minutes once daily for 4 weeks, showed significant therapeutic effects on COPD within the prescribed dosage range. It increased peak inspiratory flow, peak expiratory flow, tidal volume, and expiratory volume in rats, and also increased blood oxygen saturation.

[0024] In a rat model of acute exacerbation of chronic obstructive pulmonary disease (AECOPD), genipin-1-β-D gentiopicroside administered via nebulized inhalation for 20 minutes once daily for 4 weeks showed significant therapeutic effects within the prescribed dosage range. It increased peak inspiratory flow, peak expiratory flow, tidal volume, and expiratory volume in rats; increased blood oxygen saturation; decreased the levels of IL-6, IL-10, and TNF-α in lung tissue; and significantly reduced alveolar wall thickening, interstitial inflammatory cell infiltration, and bronchial damage.

[0025] In ammonia-induced cough model and a mouse airway phenol red excretion expectoration model, genipin-1-β-D gentiopicroside was administered via nebulized inhalation for 15 minutes each time, once a day for 3 consecutive days. Within the specified dosage range, this reduced the number of coughs induced by ammonia in mice, delayed the cough latency period, and increased the amount of airway phenol red excretion. Attached Figure Description

[0026] Figure 1 These are microscopic lung tissue pathological photographs from a COPD model experiment in rats.

[0027] Figure 2 These are microscopic lung tissue pathological photographs from rats in an AECOPD model experiment. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0029] Example:

[0030] Application of genipin-1-β-D gentiopicroside in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.

[0031] Genipin-1-β-D gentiopicroside is administered via nebulized inhalation.

[0032] Genipin-1-β-D gentiopicroside was nebulized at concentrations of 75 mg / ml and 37.5 mg / ml. After nebulization using a PARI (blue filter) nebulizer at a flow rate of 7.5 L / min for 10 min until the particle size stabilized, the aerosol particle size was measured at the nozzle outlet using a TSI particle size analyzer. Simultaneously, three parallel detection ports were selected, and the particle size values ​​at each outlet were measured at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min.

[0033] A test sample aerosol is considered stable if the particle size difference between consecutive measurements does not exceed ±20% of the mean.

[0034] The test results are shown in Table 1.

[0035] Table 1. Particle size detection results of genipin-1-β-D gentiopicroside nebulized inhalation solution

[0036]

[0037] Table 1 shows that the concentrations of genipin-1-β-D gentiopicroside in the nebulized inhalation solution were 75 mg / ml and 37.5 mg / ml, respectively. Under the condition of nebulization flow rate of 7.5 L / min, the median aerosol particle size at 0 min, 5 min, 10 min, 15 min, 20 min, 25 min and 30 min were all between 2 and 3 μm, indicating that the aerosol particle size was stable within 30 min of nebulization.

[0038] Nebulized inhalation concentration detection:

[0039]

[0040] The nebulizer flow rate was set to 7.5 L / min, and the single-port nebulizer flow rate was set to 1 L / min. Filter membrane samples were collected at seven time points: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min, after nebulization with 75 mg / ml and 37.5 mg / ml genipin-1-β-D gentiopicroside inhalation solution. The filter membranes obtained at different time points were placed in 50 ml centrifuge tubes, and 20 ml of 50% acetonitrile aqueous solution was added quantitatively. The tubes were sealed, shaken up and down, vortexed for 2 min, and then filtered through a 0.45 μm filter membrane. The filtrate was subjected to HPLC analysis under specific chromatographic conditions: an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm), a column temperature of 35℃, a mobile phase of acetonitrile-water (25:75), a flow rate of 0.6 mL / min, water as the solvent, an injection volume of 10 μL, and a run time of 10 min. The peak area obtained from the HPLC analysis was substituted into the standard curve to calculate the drug concentration in the filter membrane. The detection limit was calculated as the drug concentration in the filter membrane multiplied by 20. The detection results are shown in Table 2.

[0041] Table 2. Detection levels of genipin-1-β-D gentioside at different nebulization time points.

[0042]

[0043]

[0044] Table 2 shows that, for 75 mg / ml and 37.5 mg / ml genipin-1-β-D gentiopicroside nebulized inhalation solutions at nebulizer flow rates of 7.5 L / min and 1 L / min for single-port nebulizers, after nebulization for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min, the filter membrane concentration of 75 mg / ml genipin-1-β-D gentiopicroside remained stable at 594.133–615.776 μg / ml, and the filter membrane concentration of 37.5 mg / ml genipin-1-β-D gentiopicroside remained stable at 238.814–271.001 μg / ml, indicating that the content of genipin-1-β-D gentiopicroside remained stable during nebulization. The average membrane detection amount of genipin-1-β-D gentioside at 75 mg / ml was 12095.041 μg, and the average membrane detection amount of genipin-1-β-D gentioside at 37.5 mg / ml was 5159.191 μg. The calculated nebulized inhalation concentrations of genipin-1-β-D gentioside for the two test drugs were 2.419 mg / L and 1.032 mg / L, respectively.

[0045] Therefore, during the pharmacodynamic study, the administration method used two concentrations of genipin-1-β-D gentiopicroside nebulized inhalation solution: 75 mg / ml and 37.5 mg / ml. The nebulization flow rate was 7.5 L / min, and the nebulization inhalation time was 20 min, once daily for 4 weeks.

[0046] The actual dosage is calculated based on the nebulized inhalation concentration, nebulization time, and animal weight using the following formula:

[0047] DD(mg / kg)=C×RMV×D(×IF) / BW

[0048] Note: C is the nebulized inhalation concentration (mg / L); RMV is the inhalation volume per minute (L / min); D is the nebulization time; (×IF) is the proportion of particles in the drug solution that can enter the respiratory tract, which is generally not calculated; BW is the animal's body weight (kg); RMV (L / min) = 0.608 × BW0.852

[0049] Calculations showed that after nebulizing genipin-1-β-D gentiopicroside at concentrations of 75 mg / ml and 37.5 mg / ml for 20 min, the actual doses of genipin-1-β-D gentiopicroside administered to rats were 37.53 mg / kg and 16.01 mg / kg, respectively.

[0050] The pharmacodynamic studies of genipin-1-β-D gentiopicroside are as follows:

[0051] Experiment 1: Effect of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease (COPD).

[0052] 1.1 Test Sample:

[0053] Genipin-1-β-D Gentianoside Nebulized Inhalation Solution, Batch No.: 231212; Specification: 5ml:375mg; Production Date: 2023.12.27; Shelf Life: Tentatively 24 months; Indications: Chronic obstructive pulmonary disease (remission or acute exacerbation); Provided by: Yingkerui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0054] 1.2 Positive control drug:

[0055] Ipratropium bromide inhalation solution, batch number: 3982210, production date: 20230719; expiry date: 202506, specification: 2ml:0.5mg; dosage: one single-dose vial each time. Indication: This product is used as a bronchodilator for chronic obstructive pulmonary disease. Manufacturer: Boehringer Ingelheim (China) Investment Co., Ltd.

[0056] 1.2 Experimental animals:

[0057] 80 Wistar rats, SPF grade, weighing 130 - 150 g, with an equal number of males and females, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal license number: SCXK(Beijing)2021 - 0006, certificate number: ♀No.110011231112531117, ♂No.110011231112531048.

[0058] 1.3 Test reagents:

[0059] Table 3 Test reagents

[0060] Reagent Name Manufacturer batch number Reagent Uses 4% paraformaldehyde fixative Beyotime 0216232306 Lung tissue preservation Isoflurane Qingdao Aubofang Pharmaceutical Technology Co., Ltd. 20220306 anaesthetization Lipopolysaccharide (LPS) biosharp 22039849 Modeling

[0061] 1.4 Test instruments:

[0062] Table 4 Test instruments

[0063]

[0064]

[0065] 1.5 Dosage of the test drug:

[0066] Table 5 Test drug

[0067]

[0068] 1.6 Dosage of the positive control drug:

[0069] Table 6 Positive control drug

[0070]

[0071] 1.7 Test grouping and drug administration method:

[0072] Eighty Wistar rats, SPF grade, weighing 130–150 g, half male and half female, were used. Ten rats were randomly selected according to weight class to serve as the normal control group. The remaining rats were exposed to Huangguoshu cigarette smoke for the first 6 days of each week, twice a day for 1 hour each time, for 6 consecutive weeks. During the smoke exposure period, on the 7th day of each week for the first 4 weeks, the rats were lightly anesthetized with isoflurane and then instilled with tar (45 μL / rat) via tracheal infusion. For the next 2 weeks, the rats were instilled with LPS (100 μg / rat) via tracheal infusion. The normal control group received nebulized inhalation or tracheal infusion of physiological saline under the same conditions. In the 3rd week of modeling (day 15 of modeling), the model rats were evenly divided according to weight into a model control group, an ipratropium bromide control group, and two dosage groups: genipin-1-β-D gentiopicroside nebulized inhalation solution (75 mg / ml and 37.5 mg / ml), with 11–12 rats in each group. After grouping, each treatment group began nebulized administration once daily for 20 minutes, for 4 consecutive weeks. The normal control group and the model control group received physiological water nebulization under the same conditions. Some rats died during the modeling period, resulting in a final count of 10 rats per group. After administration, the rats were dissected and their tissues collected. Detection indicators:

[0073] ①During the administration period, the rats' body weight was measured weekly, and the results were statistically analyzed using the t-test for intergroup comparison;

[0074] ② Two weeks after administration, lung function was tested weekly, including four indicators: peak inspiratory flow, peak expiratory flow, tidal volume, and expiratory volume; the results were statistically analyzed using the t-test for intergroup comparison.

[0075] ③ After 2 weeks of drug administration, blood oxygen saturation was measured weekly; the results were statistically analyzed using the t-test for intergroup comparison.

[0076] ④ After the administration was completed, the left lung lobe of the rat was dissected for lung tissue pathological examination, the lung lesions were recorded, and statistical analysis was performed using SPSS 20.0 statistical software.

[0077] 1.8 Test Results:

[0078] Table 7 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0079]

[0080] Note: Compared with the normal control group # P<0.05, ## P<0.01

[0081] Table 7 shows that the rat body weight of the model control group was significantly reduced, which was significantly different from that of the normal control group (P<0.01); the rat body weight of the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiopicroside showed an increasing trend after 4 weeks of administration, but there was no significant difference compared with the model control group.

[0082] Table 8. Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on peak inspiratory flow rate in a rat model of chronic obstructive pulmonary disease.

[0083]

[0084]

[0085] Note: Compared with the normal control group # P<0.05; compared with the model control group* * P<0.01 * P<0.05

[0086] Table 8 shows that the peak inspiratory flow rate of rats in the model control group was significantly reduced, which was significantly different from that of the normal control group (P<0.05); the peak inspiratory flow rate of rats in the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiopicroside increased after 4 weeks of administration, which was significantly different from that of the model control group (P<0.05).

[0087] Table 9. Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on peak expiratory flow rate in a rat model of chronic obstructive pulmonary disease.

[0088]

[0089] Note: Compared with the normal control group # P<0.05; compared with the model control group* * P<0.01 * P<0.05

[0090] Table 9 shows that the peak expiratory flow rate of rats in the model control group was significantly reduced, which was significantly different from that of the normal control group (P<0.05); the peak expiratory flow rate of rats in the 75 mg / ml genipin-1-β-D gentiopicroside group increased after 3 weeks of administration and the peak expiratory flow rate of rats in the 37.5 mg / ml genipin-1-β-D gentiopicroside group increased after 4 weeks of administration, which was significantly different from that of the model control group (P<0.05).

[0091] Table 10 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on tidal volume in a rat model of chronic obstructive pulmonary disease.

[0092]

[0093] Note: Compared with the normal control group # P<0.05; compared with the model control group* * P<0.01 * P<0.05

[0094] Table 10 shows that the tidal volume of rats in the model control group was decreased, which was significantly different from that in the normal control group (P<0.05); the tidal volume of rats in the 75 mg / ml genipin-1-β-D gentiopicroside group increased after 4 weeks of administration, which was significantly different from that in the model control group (P<0.05).

[0095] Table 11 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on expiratory volume in a rat model of chronic obstructive pulmonary disease.

[0096]

[0097] Note: Compared with the normal control group # P<0.05; compared with the model control group* * P<0.01 * P<0.05

[0098] Table 11 shows that the expiratory volume of rats in the model control group was decreased, which was significantly different from that in the normal control group (P<0.05); the expiratory volume of rats in the 75 mg / ml dose group of genipin-1-β-D gentiopicroside increased after 4 weeks of administration, which was significantly different from that in the model control group (P<0.05).

[0099] Table 12 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0100]

[0101] Note: Compared with the normal control group ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0102] Table 12 shows that the blood oxygen saturation of rats in the model control group was significantly reduced, which was significantly different from that of the normal control group (P<0.01); the 75 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased the blood oxygen saturation of rats after 2 weeks of administration and the 37.5 mg / ml dose after 3 weeks of administration, which were significantly different from those of the model control group (P<0.01).

[0103] Effects on pathological changes in rat lung tissue:

[0104] Rat lung tissue was fixed in 4% paraformaldehyde fixative. After 48 hours, it was removed, rinsed with running water, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), mounted with neutral resin, and observed and photographed under a microscope. The results are shown below:

[0105] Table 13 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0106]

[0107] Note: Compared with the normal control group, ##P<0.01

[0108] Table 14 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0109]

[0110] Note: Compared with the normal control group ## P<0.01

[0111] Table 15 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0112]

[0113] Note: Compared with the normal control group ## P<0.01

[0114] Table 16 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of chronic obstructive pulmonary disease.

[0115]

[0116]

[0117] Note: Compared with the normal control group ## P<0.01

[0118] In Tables 13-16, "-" indicates normal lung parenchymal structure, no thickening, no or occasional inflammatory cells, and normal bronchial structure. "+" indicates 5-25% alveolar damage, slight thickening, slight inflammatory cell infiltration, bronchial epithelial cell cilia are bent and disordered, and some cells are detached. "++" indicates 25-50% alveolar damage, mild thickening, mild inflammatory cell infiltration, swelling and degeneration of some bronchial epithelium, and mild inflammatory exudate in the lumen. "+++" indicates 50-75% alveolar damage, moderate thickening, moderate inflammatory cell infiltration, large areas of bronchial epithelium swelling and degeneration, some epithelial cells detach, and moderate inflammatory exudate in the lumen. "++++" indicates 75-100% alveolar damage, severe thickening, severe inflammatory cell infiltration, almost complete necrosis and detachment of bronchial epithelium, and a large amount of inflammatory exudate in the lumen.

[0119] The results of Tables 13 to 16 show that: Normal control group: The lung tissue structure of rats was intact and clear, the alveolar cavity was not enlarged, there was no infiltration of inflammatory cells in the interstitium, the bronchial epithelial cells were arranged neatly, and the cilia were not damaged or shed. Model control group: There was an increase in alveolar exudation in rats, mild or moderate thickening of the alveolar wall, congestion and edema in the interstitium, and a large number of inflammatory cell infiltrations were visible, including lymphocytes, macrophages and neutrophils. The bronchial epithelium was mildly or moderately swollen and degenerated, goblet cells increased, some epithelial cells exfoliated, and there was more inflammatory exudate inside. There were significant differences in the degree of lesions compared with the normal control group. Ipratropium bromide group, 75 mg / ml group of genipin-1-β-D-gentiobioside: There was no obvious difference in the lung tissue injury of rats compared with the model control group. 37.5 mg / ml group of genipin-1-β-D-gentiobioside: The infiltration of inflammatory cells in the lung interstitium was alleviated compared with the model control group, and there were no obvious differences in alveolitis lesions, alveolar wall thickening and bronchial injury.

[0120] Experiment 2. Therapeutic effect experiment of genipin-1-β-D-gentiobioside aerosol inhalation solution on rats with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) model.

[0121] 2.1 Tested samples:

[0122] Genipin-1-β-D-gentiobioside aerosol inhalation solution, batch number: 240304; specification: 5 ml: 375 mg; production date: 20240304; validity period: tentatively 24 months; functions and indications: chronic obstructive pulmonary disease (remission or acute exacerbation period); provided by Yingkrui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0123] 2.2 Positive control drug:

[0124] Ipratropium bromide solution for inhalation, batch number: 3982215, production date: 20230909; validity period: 202508, specification: 2 ml: 0.5 mg; usage and dosage: 1 single-dose vial each time. Indications: This product is used as a bronchodilator for chronic obstructive pulmonary disease. Manufacturer: Boehringer Ingelheim (China) Investment Co., Ltd.

[0125] 2.3 Experimental animals:

[0126] 70 Wistar rats, SPF grade, 130 - 150 g, half male and half female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license number: SCXK (Beijing) 2021 - 0006, certificate number: ♀No.110011241102885668, ♂No.110011241102885563.

[0127] 2.4 Bacterial strain:

[0128] Pseudomonas aeruginosa (ATCC: VR-27853), from the ATCC Biofilm Collection Center in the United States, was passaged in our laboratory and stored at -80°C for future use.

[0129] 2.5 Test Reagents:

[0130] Table 17 Test Reagents

[0131] Reagent Name Manufacturer batch number Reagent Uses 4% tissue cell fixative Solarbio 240003008 Lung tissue preservation Isoflurane Qingdao Aubofang Pharmaceutical Technology Co., Ltd. 20220306 anaesthetization Lipopolysaccharide (LPS) SIGMA 0000135218 Modeling

[0132] 2.6 Test Instruments:

[0133] Table 18 Test Instruments

[0134]

[0135] 2.7 Dosage of the test drug:

[0136] Table 19 Test Drugs

[0137]

[0138] 2.8 Positive control drug dosage:

[0139] Table 20 Positive Control Drugs

[0140]

[0141]

[0142] 2.9 Experimental grouping and administration method:

[0143] Seventy Wistar rats, SPF grade, weighing 130–150 g, half male and half female, were used. Ten rats were randomly selected according to weight class as the normal control group. The remaining rats were smoked with Huangguoshu cigarettes twice a day for 1 hour each time for 6 weeks. LPS (200 μg / rat) was administered intratracheally on days 1, 14, and 28 of the smoke exposure (no smoke exposure was given on the day of intratracheal instillation) to establish a chronic obstructive pulmonary disease (COPD) model. On week 3 of modeling, the model rats were evenly divided according to weight into a model control group, an ipratropium bromide control group, and two dosage groups: genipin-1-β-D gentiopicroside nebulized inhalation solution 75 mg / ml and 37.5 mg / ml, with 10 rats in each group. After grouping, each treatment group began nebulized administration once a day for 20 minutes each time for 4 weeks. The normal control group and the model control group received nebulized water for injection under the same conditions. On day 39 of modeling (3 days before the end of the experiment), 6 × 10⁶ LPS was administered intratracheally. 8 CFU / ml *Pseudomonas aeruginosa* bacterial suspension, 200 μL / animal, was used to establish an acute exacerbation model of chronic obstructive pulmonary disease (COPD). Normal controls were administered saline via nebulization or intratracheal infusion under the same conditions. Organisms were dissected and tissue samples were collected after the drug administration was completed. Detection indicators:

[0144] ①During the administration period, the rats' body weight was measured weekly, and the results were statistically analyzed using the t-test for intergroup comparison;

[0145] ② Lung function was examined 2 weeks and 4 weeks after drug administration, including four indicators: peak inspiratory flow, peak expiratory flow, tidal volume, and expiratory volume. The results were statistically analyzed using the t-test for intergroup comparison.

[0146] ③ Blood oxygen saturation was measured 2 weeks and 4 weeks after drug administration, and the results were statistically analyzed using the t-test for intergroup comparison.

[0147] ④ At the end of the experiment, the right lung lobe of the rat was taken to detect inflammatory factors IL-6, IL-10 and TNF-α. The results were statistically analyzed using t-test.

[0148] ⑤ The left lung lobe of the rat was taken for lung tissue pathological examination, the lung lesions were recorded, and statistical analysis was performed using SPSS 20.0 statistical software;

[0149] ⑥ The right lung lobe of the rat was taken for proteomics analysis.

[0150] 2.10 Test Results:

[0151] Table 21 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0152]

[0153]

[0154] Note: Compared with the normal control group # P<0.05, ## P<0.01

[0155] Table 21 shows that the body weight of rats in the model control group was significantly reduced, with a significant difference compared with the normal control group (P<0.05, P<0.01); the two dose groups of genipin-1-β-D gentiopicroside, 75 mg / ml and 37.5 mg / ml, had no significant effect on the body weight of rats.

[0156] Table 22 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on peak inspiratory flow rate in a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0157]

[0158] Note: Compared with the normal control group # P<0.05, ## P<0.01; compared with the model control group *P<0.05

[0159] Table 22 shows that the peak inspiratory flow rate of rats in the model control group was significantly reduced, and there were significant differences compared with the normal control group (P<0.05, P<0.01); the 37.5 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased the peak inspiratory flow rate of rats after 2 weeks of administration, and there were significant differences compared with the model control group (P<0.05).

[0160] Table 23 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on peak expiratory flow rate in a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0161]

[0162]

[0163] Note: Compared with the normal control group # P<0.05, ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0164] Table 23 shows that the peak expiratory flow rate of rats in the model control group was significantly reduced, and there were significant differences compared with the normal control group (P<0.05, P<0.01); the 37.5 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased the peak expiratory flow rate of rats after 2 weeks of administration, and there were significant differences compared with the model control group (P<0.01).

[0165] Table 24 Effects of genipin-1-β-D gentiopicroside nebulized inhalation solution on tidal volume in a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0166]

[0167] Note: Compared with the normal control group ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0168] Table 24 shows that the tidal volume of rats in the model control group was significantly reduced, with significant differences compared to the normal control group (P<0.01). The 75 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased the tidal volume of rats after 2 weeks of administration, and the 37.5 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased the tidal volume after 2 weeks and 4 weeks of administration, with significant differences compared to the model control group (P<0.05, P<0.01). Table 25 shows the effect of genipin-1-β-D gentiopicroside nebulized inhalation solution on expiratory volume in a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0169]

[0170]

[0171] Note: Compared with the normal control group ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0172] Table 25 shows that the expiratory volume of rats in the model control group was significantly reduced, with significant differences compared to the normal control group (P<0.01). The 75 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased expiratory volume in rats after 2 weeks of administration, and the 37.5 mg / ml dose of genipin-1-β-D gentiopicroside significantly increased expiratory volume after 2 and 4 weeks of administration, with significant differences compared to the model control group (P<0.05, P<0.01). Table 26 shows the effect of genipin-1-β-D gentiopicroside nebulized inhalation solution on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0173]

[0174] Note: Compared with the normal control group ## P<0.01; compared with the model control group * P<0.05, ** P<0.01

[0175] Table 26 shows that the blood oxygen saturation of rats in the model control group was significantly decreased, with significant differences compared to the normal control group (P<0.01). The 75 mg / ml genipin-1-β-D gentiopicroside group significantly increased blood oxygen saturation in rats after 2 weeks of administration, and the 37.5 mg / ml genipin-1-β-D gentiopicroside group significantly increased blood oxygen saturation after 2 and 4 weeks of administration, with significant differences compared to the model control group (P<0.01). Table 27 shows the effect of genipin-1-β-D gentiopicroside preparation on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0176]

[0177] Note: Compared with the normal control group ## P<0.01; compared with the model control group* * P<0.01

[0178] Table 27 shows that after modeling, the levels of IL-6, IL-10, and TNF-α in the lung tissue of rats in the model control group were significantly increased, showing significant differences compared with the normal control group (P<0.01); the 75 mg / ml and 37.5 mg / ml dose groups of genipin-1-β-D gentiopicroside significantly reduced the levels of IL-6, IL-10, and TNF-α in rats after 4 weeks of administration, showing significant differences compared with the model control group (P<0.01).

[0179] Effects on pathological changes in rat lung tissue:

[0180] Rat lung tissue was fixed in 4% paraformaldehyde fixative. After 48 hours, it was removed, rinsed with running water, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), mounted with neutral resin, and observed and photographed under a microscope. The results are shown below:

[0181] Table 28 Effects of genipin-1-β-D gentiopicroside preparation on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0182]

[0183] Note: Compared with the normal control group ## P<0.01

[0184] Table 29 Effects of genipin-1-β-D gentiopicroside preparation on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0185]

[0186] Note: Compared with the normal control group ## P<0.01, compared with the model control group* * P<0.01

[0187] Table 30 Effects of genipin-1-β-D gentiopicroside preparation on a rat model of acute exacerbation of chronic obstructive pulmonary disease.

[0188]

[0189]

[0190] Note: Compared with the normal control group ##P<0.01, compared with the model control group * P<0.05 Table 31 Effects of genipin-1-β-D gentiopicroside preparation on rat model of acute exacerbation of chronic obstructive pulmonary disease

[0191]

[0192] Note: Compared with the normal control group ## P<0.01, compared with the model control group* * P<0.01

[0193] In Tables 28-31, “-” indicates normal lung parenchymal structure, no thickening, no or occasional inflammatory cells, and normal bronchial structure. “+” indicates 5-25% alveolar damage, slight thickening, slight inflammatory cell infiltration, bronchial epithelial cell cilia are bent and disordered, and some cells are detached. “++” indicates 25-50% alveolar damage, mild thickening, mild inflammatory cell infiltration, some bronchial epithelium is swollen and degenerated, and mild inflammatory exudate is visible in the lumen. “+++” indicates 50-75% alveolar damage, moderate thickening, moderate inflammatory cell infiltration, large areas of bronchial epithelium are swollen and degenerated, some epithelial cells are detached, and moderate inflammatory exudate is visible in the lumen. “++++” indicates 75-100% alveolar damage, severe thickening, severe inflammatory cell infiltration, almost complete necrosis and detachment of bronchial epithelium, and a large amount of inflammatory exudate is visible in the lumen.

[0194] Tables 28-31 show the results: In the normal control group, the lung tissue structure of rats was clear, with no thickening of the alveolar walls, no interstitial congestion, edema, or inflammatory cell infiltration, and the bronchial epithelial cells were neatly arranged without swelling, degeneration, or necrosis. After modeling, in the model control group, the alveolar walls of rats were mildly or moderately thickened, with interstitial congestion and edema, and fibroblast proliferation; a large number of neutrophils, lymphocytes, and mononuclear macrophages were infiltrated around blood vessels, bronchi, and alveolar septa, and granulomas or abscesses were observed in some areas; there was mild inflammatory cell exudation in the alveolar cavities; the bronchial epithelium was mildly to severely swollen and degenerated, with some epithelial cells sloughing off; and a large amount of inflammatory exudate was observed in the bronchial lumen. The degree of lesions was significantly different from that of the normal control group (P<0.01).

[0195] Ipratropium bromide group: Alveolar wall thickening and bronchial damage in rats were slightly reduced compared to the model control group. Genipin-1-β-D gentiopicroside 75 mg / mL group: Alveolar wall thickening, interstitial inflammatory cell infiltration, and bronchial damage in rats were slightly reduced compared to the model control group. Genipin-1-β-D gentiopicroside 37.5 mg / mL group: Mild or slight thickening of alveolar walls was observed in rats; mild to moderate interstitial inflammatory cell infiltration was observed; bronchial epithelial cells showed mild to moderate swelling and degeneration. The lesion grading was significantly reduced compared to the model control group.

[0196] Experiment 3: Antitussive effect test of genipin-1-β-D-gentiobioside aerosol inhalation solution on the cough model of mice induced by ammonia water.

[0197] 3.1 Tested drug:

[0198] Genipin-1-β-D-gentiobioside aerosol inhalation solution, batch number: 230608; production date: 20230630; specification: 5 ml: 375 mg; expiration date: tentatively 24 months; functions and indications: chronic obstructive pulmonary disease (remission or acute exacerbation phase); provided by Yingke Rui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0199] 3.2 Positive control drug:

[0200] Budesonide suspension for inhalation, batch number: 329723, production date: 2023.08, expiration date: 2025.07. Produced by AstraZeneca Pharmaceutical Co., Ltd. Composition: The active ingredient of this product is budesonide. Character: A suspension of fine particles. Indications: Treatment of bronchial asthma. Specification: 1 mg: 2 ml. Dosage and administration: Aerosol inhalation, 1 - 2 mg / time, twice a day.

[0201] 3.3 Test animals:

[0202] 50 ICR rats, SPF grade, 18 - 22 g, half male and half female, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license number: SCXK(Beijing)2021 - 0006, certificate number: 110324241101008338.

[0203] 3.4 Test instruments:

[0204] Table 32 Test instruments

[0205]

[0206] 3.5 Dosage of tested drug:

[0207] The dosages of genipin-1-β-D-gentiobioside for mice are two dose groups of 75 mg / ml / d and 37.5 mg / ml / d. Aerosol inhalation administration, once a day, 15 minutes each time.

[0208] 3.6 Dosage of positive control drug:

[0209] Atomize and inhale the original liquid of 1 mg: 2 ml for administration, once a day, 15 minutes each time.

[0210] 3.7 Test grouping and administration method:

[0211] (1) Screening qualified mice: ICR mice, weighing 20±2g, half male and half female, were induced to cough by ultrasonic atomization spray of 25% ammonia water for 15s. The time from the start of spraying to the appearance of abdominal muscle contraction and mouth opening was observed. Those that did not cough within 1.5min were discarded.

[0212] (2) Observation of drug effects: Qualified mice were randomly divided into a model control group, a budesonide positive control group, and genipin-1-β-D gentiopicroside dosage groups of 75 mg / ml / d and 37.5 mg / ml / d, with 10 mice in each group, half male and half female. Each treatment group received nebulized medication for 15 minutes, once daily for 3 consecutive days. The model control group received physiological saline under the same conditions. One hour after administration on the 3rd day, each mouse was induced to cough for 15 seconds by ultrasonic nebulization with 25% ammonia water. Coughing was measured by the contraction of the mouse's abdominal muscles or chest, and simultaneous opening of the mouth. The latency period from the start of nebulization to the onset of cough and the number of coughs within 3 minutes were observed in each group. The results were statistically analyzed.

[0213] 3.8 Test Results:

[0214] Table 33. Antitussive effect of genipin-1-β-D gentiopicroside nebulized inhalation solution on ammonia-induced cough model in mice.

[0215]

[0216] Compared with the model control group, **P<0.01, *P<0.05

[0217] Table 33 shows that the 75 mg / ml dose of genipin-1-β-D gentiopicroside significantly prolonged the latency period of ammonia-induced cough in mice, while the 37.5 mg / ml dose significantly reduced the number of coughs induced by ammonia and prolonged the latency period of ammonia-induced cough in mice, showing significant differences compared with the model group (P<0.05, P<0.01).

[0218] Experiment 4: Expectorant effect of genipin-1-β-D gentiopicroside nebulized inhalation solution on phenol red excretion in mouse airways.

[0219] 4.1 Test drug:

[0220] Genipin-1-β-D Gentianoside Nebulized Inhalation Solution, Batch No.: 230608; Specification: 5ml: 375mg; Shelf life: tentatively 24 months; Indications: Chronic obstructive pulmonary disease (remission or acute exacerbation); Provided by: Yingkerui (Tianjin) Innovative Pharmaceutical Research Co., Ltd.

[0221] 4.2 Positive control drug:

[0222] Acetylcysteine Solution for Inhalation: Batch number: 28006666, Production date: May 2023, Expiry date: April 2028. Produced by Zambon Pharmaceutical Co., Ltd. Ingredients: The active ingredient of this product is acetylcysteine. Appearance: A clear liquid that is colorless or slightly light blue-violet, with a slight sulfur smell. Indications: Treat respiratory diseases with excessive thick mucus secretions. Specification: 3 ml / ampoule. Dosage and administration: By nebulization inhalation, 3 ml each time, 1 - 2 times a day.

[0223] 4.3 Test animals:

[0224] 50 ICR rats, SPF grade, weighing 18 - 22 g, with an equal number of males and females, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Animal License Number: SCXK(Beijing)2021 - 0006, Certificate Number: 110324241101007874.

[0225] 4.4 Test instruments:

[0226] Table 34 Test instruments

[0227]

[0228] 4.5 Dosage of the test drug:

[0229] For genipin - 1 - β - D - gentiobioside in mice, there are two dosage groups: 75 mg / ml / d and 37.5 mg / ml / d. Administered by nebulization inhalation, once a day, for 15 minutes each time.

[0230] 4.6 Dosage of the positive control drug:

[0231] Acetylcysteine Solution for Inhalation, the original drug solution is nebulized and inhaled, once a day, for 15 minutes each time.

[0232] 4.7 Test grouping and administration method:

[0233] Forty ICR mice, weighing 18-22g (half male and half female), were randomly divided into four groups according to body weight: a model control group, an acetylcysteine ​​group, and two dosage groups (75mg / kg / d and 37.5mg / kg / d) of genipin-1-β-D gentiopicroside, with 10 mice in each group. All treatment groups received the medication via nebulization for 15 minutes, once daily for three consecutive days. The model control group received physiological saline under the same conditions. Thirty minutes after administration on day 3, each mouse was intraperitoneally injected with a 2.5% phenol red saline solution (250 mg / kg). Thirty minutes later, the mice were euthanized by cervical dislocation. The peritracheal tissue was dissected, and a section of the trachea from the thyroid cartilage to the tracheal branch was cut off and placed in a test tube containing 1 ml of saline. Then, 0.1 ml of 1 mol / L NaOH solution was added, and the test tube was stored in a refrigerator. When measuring the OD value, the solution in the entire test tube was brought to a final volume of 2 ml with saline. The tube was centrifuged at 2000 r / min for 8 min, and the supernatant was collected. The absorbance value at 559 nm was measured using an ELISA reader. The amount of phenol red excreted in the respiratory tract of each mouse was calculated according to the standard curve equation, and the amount of phenol red excreted in each group of mice was compared, i.e., the amount of sputum produced.

[0234] Construction of the phenol red standard curve: Weigh a certain amount of phenol red and dissolve it in 1 mol / L NaOH solution to make the concentration of phenol red 1.00 mg / ml. Dilute the solution with 1 mol / L NaOH to obtain concentrations of 20.0, 10.0, 5.00, 2.50, 1.25, 0.625, 0.3125, and 0.1562 μg / ml, and measure the absorbance values. Plot the standard curve with concentration on the x-axis and absorbance values ​​on the y-axis.

[0235] 4.8 Test Results:

[0236] Table 35. Expectorant effect of genipin-1-β-D gentiopicroside on a mouse phenol red excretion model.

[0237]

[0238] Compared with the model control group, **P<0.01

[0239] Table 35 shows that both the 75 mg / kg / d and 37.5 mg / kg / d doses of genipin-1-β-D gentiopicroside significantly increased the amount of phenol red excreted from the trachea of ​​mice, with a significant difference compared with the model control group (P<0.01).

Claims

1. Application of genipin-1-β-D gentiopicroside in the preparation of drugs for the treatment of chronic obstructive pulmonary disease.

2. The application of genipin-1-β-D gentiopicroside according to claim 1, characterized in that: The genipin-1-β-D gentiopicroside is administered via nebulized inhalation.

3. The application of genipin-1-β-D gentiopicroside according to claim 1, characterized in that: The concentration of the nebulized inhalation solution of genipin-1-β-D gentiopicroside is 75 mg / ml or 37.5 mg / ml, the nebulization flow rate is 7.5 L / min, and the nebulization time is 0–30 min.

4. The application of genipin-1-β-D gentiopicroside according to claim 3, characterized in that: The nebulized inhalation concentration of genipin-1-β-D gentiopicroside at 75 mg / ml is 2.419 mg / L.

5. The application of genipin-1-β-D gentiopicroside according to claim 3, characterized in that: The nebulized inhalation concentration of genipin-1-β-D gentiopicroside (37.5 mg / ml) is 1.032 mg / L.

6. The application of genipin-1-β-D gentiopicroside according to claim 2, characterized in that: The nebulized inhalation solution of genipin-1-β-D gentiopicroside comprises the following raw materials: genipin-1-β-D gentiopicroside, pH adjuster, osmotic pressure adjuster, and solvent.

7. The application of genipin-1-β-D gentiopicroside according to claim 6, characterized in that: The method for preparing the nebulized inhalation solution of genipin-1-β-D gentiopicroside includes the following steps: (1) Measure out 40% to 90% of the total volume of the solvent required to prepare the solution to obtain the first solution; (2) Add an osmotic pressure regulator to the first solution and stir until homogeneous to obtain the second solution; (3) Add a pH adjuster to the second solution to adjust the pH value to 4.5-7.0 to obtain the third solution; (4) Add genipin-1-β-D gentiopicroside to the third solution, stir well, and add a pH adjuster to keep the pH value between 4.5 and 7.0 to obtain the fourth solution; (5) Add solvent to the fourth solution and bring the volume up to the required total volume of the solution to be prepared. Stir well to obtain the nebulized inhalation solution of genipin-1-β-D gentiopicroside.

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