A pharmaceutical composition for treating allergic asthma, its preparation method and application

Abstract

This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition for treating allergic asthma, its preparation method, and its application. The pharmaceutical composition for treating allergic asthma of this invention is obtained by combining luteolin, arbutin, and isopyramidal lactone in a specific ratio. The resulting pharmaceutical composition can significantly improve the pathological characteristics of asthma; it can improve pathological changes caused by asthma, such as bronchial stenosis, increased airway mucus, thickening of bronchial smooth muscle, and a large number of inflammatory cells around the airways; it can reduce lung inflammation caused by allergic asthma; it can improve airway remodeling in allergic asthma; it can reduce lung tissue apoptosis and lung damage caused by allergic asthma; it can alleviate the symptoms of allergic asthma and has a protective effect against allergic asthma. It can be used in the preparation of asthma drugs, anti-allergic inflammatory drugs, and drugs that reduce lung tissue apoptosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a pharmaceutical composition for treating allergic asthma, its preparation method, and its application. Background Technology

[0002] Asthma is a heterogeneous disease characterized by chronic airway inflammation, in which various cells interact with epithelial cells, leading to airway hyperresponsiveness, airway inflammation, and airway remodeling. According to a World Health Organization report, asthma affects approximately 339 million people worldwide, and its incidence is increasing at a rate of 20%-25% annually, projected to reach 400 million by 2025, causing a significant health and economic burden. Among asthma patients, 66-83% are caused by allergies. Allergic asthma is a chronic respiratory disease caused by a type II immune response, with a complex pathogenesis, primarily leading to T cell differentiation into Th2 cells and the production of Th2 cytokines. Simultaneously, allergic asthma is characterized by the production of type II inflammatory cytokines (IL-4, IL-5, and IL-13, etc.), airway remodeling, and airway hyperresponsiveness [10-11]. Currently, clinical treatment for allergic asthma is divided into control therapy and remission therapy. Control therapy focuses on anti-inflammation, with inhaled corticosteroids (ICS) combined with long-acting β2 receptor agonists (LABA) as the first-line drugs. Racistative therapy primarily aims to rapidly relieve asthma attack symptoms by relieving bronchospasm. This includes short-acting β2-agonists (SABA), short-acting theophylline, and inhaled anticholinergics, with SABA being the first-line drug for acistative therapy (e.g., salbutamol, terbutaline). Currently, relieving allergic asthma attacks clinically is not difficult; the challenge lies in controlling recurrent attacks. Most allergic asthma patients receive good treatment through anti-inflammatory drugs, but 5%-10% require higher doses and may develop severe asthma. Glucocorticoids, as first-line anti-inflammatory drugs for asthma, have achieved good clinical efficacy, but long-term use can lead to a series of side effects such as cataracts and tachycardia. In addition, conventional drugs such as leukotriene receptor antagonists, cholinergic antagonists, and sustained-release theophylline are used to control asthma attacks, but these conventional drugs cannot fundamentally reduce airway remodeling. These factors necessitate the search for new and effective drugs.

[0003] The main characteristics of allergic inflammatory responses are elevated levels of eosinophils, Th2 cells, Th2-driven inflammatory factors, and IgE. Studies have found that reducing IgE is one effective method for treating allergic asthma; therefore, targeting IgE may be a novel treatment option. Th2 cell cytokines (IL-4, IL-5, etc.), especially IL-13, can directly act on airway smooth muscle cells, altering their contraction and relaxation responses, making them key targets for treating allergic asthma. In addition to related cytokines, phosphatidylinositol-3-kinase (PI3K) is a downstream signaling effector of G protein-coupled receptors, while serine / threonine kinase (Akt) is a direct downstream key signal of PI3K; PI3K / Akt together form an intracellular signaling pathway. PI3K participates in chronic airway inflammation by promoting the activation of inflammatory mediators, eosinophil oligoaggregation, and Th1 / Th2 immune imbalance. It has been reported that inhibiting PI3K can effectively reduce airway inflammation in asthma.

[0004] Therefore, finding a potentially effective drug to treat allergic asthma, control the incidence of allergic asthma, alleviate allergic asthma symptoms, and protect lung tissue cells from apoptosis, is a very urgent issue. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a pharmaceutical composition for treating allergic asthma, its application, and a method for its preparation. Specifically, this is achieved through the following technical solution:

[0006] A pharmaceutical composition for treating allergic asthma comprises the following raw materials: luteolin, arbutin, and isopregan lactone. Further, the weight parts of each raw material are: 60-120 parts luteolin, 110-170 parts arbutin, and 580-640 parts isopregan lactone; a preferred ratio is 89 parts luteolin, 144 parts arbutin, and 609 parts isopregan lactone.

[0007] Furthermore, the composition consists only of luteolin, arbutin, and isopyram lactone.

[0008] The pharmaceutical composition of this application can alleviate asthma symptoms and control the incidence of allergic asthma, and can be used in the preparation of asthma drugs; it can alleviate allergy symptoms, and can be used in the preparation of anti-allergic inflammatory drugs; it can reduce lung tissue apoptosis, and can be used in the preparation of drugs that reduce lung tissue apoptosis.

[0009] The method for preparing the pharmaceutical composition for treating allergic asthma is characterized by mixing luteolin, arbutin, and isopyram lactone purified products.

[0010] Compared with the prior art, the technical effects of this invention are reflected in:

[0011] The pharmaceutical composition for treating allergic asthma of this invention is obtained by combining luteolin, arbutin, and isopyramidal lactone in a specific ratio. The resulting pharmaceutical composition can significantly improve the pathological characteristics of asthma; it can significantly reduce the levels of IgE, IL-4, IL-5, IL-13, and TNF-α, and inhibit the concentration of the pro-inflammatory cytokine TNF-α, thereby improving the mediation of Th2 cytokines; it can improve the pathological changes caused by asthma, such as bronchial stenosis, increased airway mucus, thickening of bronchial smooth muscle, and a large number of inflammatory cells around the airways; it can alleviate lung inflammation caused by allergic asthma; it can improve airway remodeling in allergic asthma; it can reduce lung tissue apoptosis and lung damage caused by allergic asthma; it can alleviate the symptoms of allergic asthma and has a protective effect against allergic asthma. It can be used in the preparation of asthma drugs, anti-allergic inflammatory drugs, and drugs to reduce lung tissue apoptosis. Furthermore, the composition of this application achieves good results with only a small dosage, and the dosage required to achieve the same effect is far lower than that of *Hedyotis diffusa* extract. Attached Figure Description

[0012] Figure 1 This is a flowchart of the establishment and drug treatment process for a mouse model of allergic asthma.

[0013] Figure 2 This is a graph showing the results of the pharmaceutical composition for treating allergic asthma in this application improving OVA-induced behavioral characteristics and reducing the levels of IgE, IL-5, IL-4, IL-13, and TNF-α in the serum of OVA-induced mice; A: Effects of different treatment methods on OVA-induced mouse behavior; B-F: Serum levels of IgE, IL-5, IL-4, IL-13, and TNF-α in mice compared to the normal group. ## P < 0.01, ### P < 0.001.

[0014] Figure 3 This is a characteristic map (H&E staining, scale bar = 50 μm) showing the improvement in lung tissue pathological changes by the pharmaceutical composition for treating allergic asthma in this application. Compared with the normal group, ### P < 0.001; compared with the OVA model group, ** P < 0.01, *** P<0.001;×400, mean±SD, n=4.

[0015] Figure 4 This is a graph showing the results of the pharmaceutical composition for treating allergic asthma in this application reducing the levels of IL-4, IL-5, and TNF-α and the number of eosinophils in bronchoalveolar lavage fluid; A-C: Levels of IL-4, IL-5, and TNF-α in bronchoalveolar lavage fluid; D: Number of eosinophils in bronchoalveolar lavage fluid, compared with the normal group. ##P < 0.01, ### P < 0.001; compared with the OVA model group, * P < 0.05, ** P < 0.01, *** P < 0.001; mean ± SD, n = 4.

[0016] Figure 5 This is a figure showing the effect of the pharmaceutical composition for treating allergic asthma in this application on the pathological changes in OVA-induced lung tissue in mice. PAS staining was used to detect goblet cells and mucus levels in lung tissue (PAS staining, scale bar = 50 μm); compared with the normal group, ### P < 0.001; compared with the OVA model group, ** P < 0.01, *** P < 0.001; × 400, mean ± SD, n = 4; (PAS: periodic acid-Schiff). 3

[0017] Figure 6 This is a figure showing the effect of the pharmaceutical composition for treating allergic asthma described in this application on the pathological changes in lung tissue induced by OVA in mice. Masson staining was used to detect collagen deposition and airway wall thickening in lung tissue (Masson staining, scale bar = 50 μm). Compared with the normal group, ### P < 0.001; compared with the OVA model group, *** P<0.001;×400, mean±SD, n=4.

[0018] Figure 7 This image shows the results of the pharmaceutical composition for treating allergic asthma in this application reducing lung tissue cell apoptosis induced by OVA. A: Typical image of lung tissue apoptosis (TUNEL staining, scale bar = 50 μm), B: Analysis of the proportion of TUNEL-positive cells; compared with the normal group, ## P < 0.01; compared with the OVA model group, * P < 0.05 ** P<0.01;×400, mean±SD, n=4.

[0019] Figure 8 This image shows the results of the pharmaceutical composition for treating allergic asthma in this application reducing lung tissue apoptosis in mice with allergic asthma. A: Immunohistochemical images of Bax, Bcl-2, and Caspase-3 in each group (scale bar = 40 μm); B: Statistical graph of Bax-positive area percentage; C: Statistical graph of Bcl-2-positive area percentage; D: Statistical graph of caspase-3-positive area percentage; Compared with the normal group, # P < 0.05 ## P < 0.01; compared with the OVA model group, *P < 0.05 ** P<0.01;×400, mean±SD, n=4.

[0020] Figure 9 These are the results of Western blot analysis of PI3K, p-PI3K, and p-Akt / Akt protein expression in lung tissue; A: Western blot images of PI3K, p-PI3K, and p-Akt / Akt; B-D: Representative images of PI3K, p-PI3K, and p-Akt / Akt protein levels in lung tissue determined by Western blot analysis; compared with the normal group. ## P < 0.01, ### P < 0.001; compared with the OVA model group, ** P < 0.01, *** P<0.001; mean±SD, n=4. Detailed Implementation

[0021] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description.

[0022] Example 1

[0023] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0024] The mixture of 89g of luteolin, 144g of arbutin, and 609g of isopyram lactone was obtained by mixing them evenly.

[0025] Example 2

[0026] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0027] The mixture of 60g of luteolin, 170g of arbutin, and 640g of isopyram lactone was obtained by mixing them evenly.

[0028] Example 3

[0029] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0030] The mixture of 120g of luteolin, 110g of arbutin, and 580g of isopyram lactone was obtained by mixing them evenly.

[0031] Example 4

[0032] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0033] The mixture of 120g of luteolin, 110g of arbutin, and 640g of isopyram lactone was obtained by mixing them evenly.

[0034] Example 5

[0035] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0036] The mixture of 60g of luteolin, 170g of arbutin, and 580g of isopyram lactone was obtained by mixing them evenly.

[0037] Example 6

[0038] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0039] The mixture of 120g of luteolin, 170g of arbutin, and 580g of isopyram lactone was obtained by mixing them evenly.

[0040] Example 7

[0041] A pharmaceutical composition for treating allergic asthma is prepared by the following steps:

[0042] The mixture of 60g of luteolin, 110g of arbutin, and 640g of isopyram lactone was obtained by mixing them evenly.

[0043] 1. Materials and Methods

[0044] 1.1 Chemicals and Reagents

[0045] Ovalbumin (OVA, SLCB8249) was purchased from Sigma-Aldrich, USA. Aluminum hydroxide adjuvant (TE267860) was purchased from Thermo Fisher Scientific, USA. Dexamethasone (DEX, H03N5S43119) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Arbutin (AF21041151, ≥98%), isopyramidalin (AF20071206, ≥98%), and luteolin (AF20030854, ≥98%) were purchased from Chengdu Efa Biotechnology Co., Ltd. (Chengdu, China). Wright's staining solution (20200910) was purchased from SolarBio. Mouse ovalbumin-specific immunoglobulin E (IgE, ZC-38496), IL-4 (ZC-37986), IL-13 (ZC-37967), and TNF-α (ZC-39024) ELISA kits were purchased from Shanghai Zhuocai Biotechnology Co., Ltd. (Shanghai, China). Bax (Bs-0127R) and Bcl-2 (Bs-20351R) were purchased from Beijing Bio-Sensing Biotechnology Co., Ltd. (Beijing, China). RIPA cell lysis buffer (20141219), protein phosphatase inhibitor (20151014), and BCA protein concentration assay kit (20140722) were purchased from SolarBio. The Western blot kit for high-sensitivity chemiluminescence detection (AC21141) was purchased from Beijing Kangwei Century Biotechnology Co., Ltd. (Beijing, China). PVDF membrane (K4SA1716L) was purchased from Millipore, USA. Caspase-3 (19677-1-AP), β-actin (66009-1-lg), goat anti-mouse IgG antibody (SA00001-1), and goat anti-rabbit IgG antibody (SA00001-2) were purchased from Proteintech, USA. Akt (ab179463), p-Akt (ab38449), PI3K (ab191606), and p-PI3K (ab182651) were purchased from Abcam, UK. All other chemical reagents were of analytical grade.

[0046] 1.2 Preparation of GPH extract

[0047] Gerbera piloselloides was purchased from Duyun City, Guizhou Province, China, and identified by Professor Zhang Xu of the School of Pharmacy, Guizhou Medical University as the dried whole herb of Gerbera piloselloides (G. piloselloides) in the Compositae family. The voucher specimen was stored in the Guizhou Provincial Key Laboratory of Pharmaceutical Preparations, Guizhou Medical University. The medicinal materials were pulverized and extracted with 10-fold, 8-fold, and 6-fold (W / V) 50% ethanol for 2 h, three times in total. The extraction solutions were combined and collected, and ethanol was recovered under reduced pressure. The extract dissolved in water was added to a pretreated D101 macroporous resin column (180405, Shanghai Lanyu Technology Development Co., Ltd.), and then washed successively with 4 column volumes of water, 60% ethanol, and 95% ethanol. Finally, the eluate containing 60% ethanol was collected and evaporated to dryness using a rotary evaporator (Rotava-tor Evolorator R200 BUCHI, Switzerland) to obtain the GPH extract (yield: 2.77%).

[0048] 1.3 Animals and experimental groups

[0049] Sixty female healthy Kunming mice (20 ± 2 g) were provided by Changsha Tianqin Biotechnology Co., Ltd. (Changsha, China). The production license number for experimental animals was: SCXK(Xiang) 2014-0011. The license number for the use of experimental animals was: SCXK(Qian) 2018-0001. All mice were housed under standard laboratory conditions for 1 week before the start of the experiment, at a temperature of (20-25 °C), a humidity of (55 ± 5%), a 12 / 12 h light / dark cycle, with free access to water and food. All animal experiments complied with the regulations on the management of experimental animals of Guizhou Medical University and the "Animal Protection Law of the People's Republic of China". After 7 days of acclimation, all mice were randomly divided into 5 groups (n = 12 / group): normal group (Con), ovalbumin group (OVA), Gerbera piloselloides extract group (GPH, 111 mg / kg), drug composition for treating allergic asthma (Mixture, arbutin:marmesin:luteolin = 1.44 mg / kg: 6.09 mg / kg: 0.89 mg / kg), dexamethasone group (DEX, 1.00 mg / kg, orally). Mice in the normal group received only an equal volume of normal saline. The schedule for the establishment and treatment of the asthma model is as Figure 1 shown.

[0050] 1.4 Establishment of the allergic asthma model

[0051] Except for the normal control group, each group received an intraperitoneal injection of 0.1 mL of sensitization solution (containing 25 μg OVA and 2 mg aluminum hydroxide adjuvant) on days 0, 7, and 14, while the normal control group received an intraperitoneal injection of the same volume of normal saline. From days 21 to 26, except for the normal control group, challenge was performed with 2% OVA (1 mL / min, 20 min), while the normal control group received normal saline instead of the challenge solution, for 6 consecutive days. Starting on day 21, 30 min before nebulization, the drug treatment groups received the corresponding drugs via gavage, while the normal control group and the model group received the same volume of normal saline.

[0052] 1.5 Behavioral Indicators

[0053] On day 26, after the nebulization was completed and the stimulating liquid on the mice's fur dried, the asthma behavior indicators (asthma attack characteristics such as scratching the nose and itching) of the mice were observed and scored within 10 minutes: 0 points for no scratching of the nose or itching, 1-3 times for scratching the nose or itching, 2 points for 4-6 times, and 3 points for 7 times or more. The behavioral evaluation of the mice was carried out according to this standard.

[0054] 1.6 Blood and bronchoalveolar lavage fluid (BALF) collection

[0055] Twenty-four hours after the final OVA challenge, mice were sacrificed, and blood was collected from the orbital cavity. The collected serum was centrifuged at 3000 rpm for 10 min at 4°C for 4°C. The serum was stored at -80°C for the detection of IgE, IL-5, IL-13, and TNF-α. BALF samples were randomly collected from four mice via endotracheal intubation. The samples were washed twice with phosphate-buffered saline (PBS, 1 ml, pH 7.2-7.4), centrifuged at 2500 rpm for 10 min at 4°C, and the supernatant was collected and stored at -20°C. IL-4, IL-5, and TNF-α were detected using an ELISA kit. The precipitated cells were resuspended in 1 ml of cold PBS buffer, stained with Wright's stain, and the total cell count and differential count were performed under a microscope. 300 stained cells were randomly selected from different fields of view, and the proportion of eosinophils was calculated.

[0056] 1.7 Histological Analysis

[0057] For histological analysis, the left lung was fixed overnight in 4% paraformaldehyde at room temperature and then embedded in paraffin for sectioning. Tissue sections (3 μm) were dewaxed with xylene. The tissue sections were stained with hematoxylin and eosin (H&E) to observe pathological changes such as alveolar structure and inflammatory cell infiltration. The specific scoring criteria are as follows: 0 = no infiltration; 1 = inflammatory infiltration of the alveolar septa, no thickening of the alveolar septa, pathological degree <25%; 2 = significant inflammatory infiltration, mild thickening of the alveolar septa, lesion degree 25%-50%; 3 = significant inflammatory infiltration, significant increase in alveolar septa, lesion degree 50%-75%; 4 = fibrosis, lesion degree >75%.

[0058] Tissue sections were stained with Schiff periodic acid (PAS) and Masson staining. Stained sections were examined under an optical microscope by pathologists in blinded groups. PAS staining was used to observe the staining of PAS-positive epithelial cells (goblet cells). The specific scoring system was as follows: 0 points, no goblet cells; 1 point, stained area less than 25%; 2 points, stained area 25%-50%; 3 points, stained area 50%-75%; 4 points, stained area greater than 75%. The thickness of the Masson-positive airway basement membrane and collagen deposition were evaluated using a semi-quantitative method. Sections from four randomly selected mice in each group were evaluated and imaged using a Pannoramic 250 digital slide scanner (3DHISTECH Hungary). 1.8TdT-mediated dUTP nick-end marker (TUNEL) staining.

[0059] Left lung tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4 μm). TUNEL staining was performed using terminal deoxynucleotidyl transferase (TdT)-mediated fluorescent dUTP nick-end labeling (TUNEL). TUNEL staining was performed according to the TUNEL kit instructions (49330900, Roche Group, Swit). Apoptotic cell nuclei appeared green. The apoptosis index was calculated from three randomly selected fields of view under a high-power microscope (×400).

[0060] 1.9 Immunohistochemical analysis

[0061] The expression of Bcl-2, Bax, and Caspase 3 in paraffin sections was detected by immunohistochemistry. Endogenous peroxidase was blocked with 3% H2O2, followed by heating to 97℃ for 20 min in antigen retrieval solution. The sections were incubated with goat serum blocking solution (SP9001, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) at room temperature for 20 min, and then incubated overnight at 4℃ with anti-Bcl-2 (1:100), anti-Bax (1:200), and anti-Caspase-3 (1:200). After incubation with primary antibody, the sections were washed three times with PBS (pH=7.4), incubated with secondary antibody, stained with 3,3-diaminobenzidine (DAB) (1:20), and reverse-stained with hematoxylin. The staining was visualized under a microscope to assess the degree of cellular immunopositivity. Hematoxylin staining showed blue nuclei, while DAB-positive expression appeared brownish-yellow. 1.10 Western blotting

[0062] Total protein was extracted from right lung tissue of mice using radioimmunoprecipitation (RIPA) lysis buffer, and its concentration was determined using a BCA protein assay kit. 30 μg of protein sample was subjected to SDS-PAGE (8% sodium dodecyl sulfate polyacrylamide gel electrophoresis). Subsequently, the protein was electrotransferred to a PVDF membrane, blocked with 5% BSA, and incubated overnight at 4°C with primary antibodies Akt (1:10000), p-Akt (1:800), PI3K (1:1000), p-PI3K (1:1000), and β-actin (1:5000), respectively. The membrane was then washed and incubated at room temperature for 2 h with goat anti-mouse secondary antibody (1:5000) and goat anti-rabbit secondary antibody (1:5000). After washing with TBST, ECL reagent was added, and the membrane was allowed to react fully with the chromogenic solution for 1 min. The membrane was then exposed to light using a gel imaging system (Bio-Rad Laboratories, USA), and photographed for storage. The experimental results were analyzed using the Quantity One gel imaging system to determine the grayscale values ​​of the target protein and the internal control protein. The experiment was repeated three times, with β-actin as the internal control.

[0063] 1.11 Statistical Analysis

[0064] Statistical analysis was performed using SPSS 20.0 statistical software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software, Inc., USA). Results are expressed as mean ± SD. Data analysis was performed using t-tests and one-way ANOVA. Differences were considered statistically significant (p < 0.05) after a two-tailed t-test.

[0065] 2. Results

[0066] 2.1 Behavioral Changes

[0067] Clinical symptoms of allergic asthma often manifest as cough, wheezing, and dyspnea. However, in mice, these symptoms include cyanosis of the lips, scratching of the nose, rapid breathing, significant abdominal distension, and restlessness. Compared to the control group, OVA-induced asthma behavior scores in mice were significantly increased, while those in mice treated with GPH, Mixture, or DEX showed significantly decreased scores. This result indicates that Mixture can significantly improve the pathological features of OVA-induced asthma in mice, such as... Figure 2 As shown in Figure A.

[0068] 2.2 Effects of Mixture on Serum Th1 / Th2 Cytokines and Serum OVA-Specific IgE Levels

[0069] The levels of Th2 cytokines IL-4, IL-5, and IL-13, as well as pro-inflammatory cytokines TNF-α and IgE in serum were detected using enzyme-linked immunosorbent assay (ELISA) to evaluate the Th1 / Th2 immune imbalance. Figure 2 As shown in the BF diagram, compared with the Con group, OVA sensitization and challenge significantly increased the serum levels of IgE, IL-4, IL-5, IL-13, and TNF-α in mice. Compared with the OVA group, GPH, Mixture, or DEX significantly decreased the levels of IgE, IL-4, IL-5, IL-13, and TNF-α. These data suggest that Mixture may inhibit the concentration of the pro-inflammatory cytokine TNF-α, thereby improving the mediation of Th2 cytokines.

[0070] 2.3 Mixture reduces lung inflammation in mice with allergic asthma

[0071] Observe the pathological changes in lung tissue of each group ( Figure 3 H&E staining results showed that the bronchial and lung tissue structures of mice in the Con group were intact, the thickness of bronchial smooth muscle was normal, no inflammatory cell infiltration was observed around the bronchi, and the alveoli were of normal size. OVA-induced allergic asthma mice showed bronchial stenosis, increased airway mucus, thickened bronchial smooth muscle, and a large number of inflammatory cells around the airways. GPH, Mixture, or DEX significantly improved these pathological changes. Evaluation analysis showed that the degree of lung tissue lesions in the OVA group was significantly higher than that in the Con group, while the degree of lung tissue lesions in the GPH, Mixture, or DEX groups was significantly lower than that in the OVA group.

[0072] Then, the levels of Th2 cytokines and the pro-inflammatory factor TNF-α in the BALF of mice in each group were measured. Figure 4 AC). Results showed that the expression levels of Th2 cytokines IL-4 and IL-5 and the pro-inflammatory cytokine TNF-α in the OVA group were significantly higher than those in the Con group. The GPH, Mixture, or DEX groups significantly reduced the expression levels of IL-4, IL-5, and TNF-α. Simultaneously, inflammatory cells in the BALF of mice in each group were counted. Figure 4 (D) The results showed that the total number of eosinophils was significantly increased in the OVA group compared with the Con group. The total number of eosinophils was significantly decreased in the GPH, Mixture, or DEX groups compared with the OVA group. These results suggest that Mixture may alleviate lung inflammation caused by allergic asthma.

[0073] 2.4 Mixture improves airway remodeling in OVA-induced allergic asthma mice

[0074] Goblet cell proliferation, collagen deposition, and airway wall thickening are key pathological changes in asthma and are often used to assess the severity of airway remodeling. In this study, goblet cell proliferation was assessed using periodic acid Schiff (PAS) staining. Figure 5 As shown, compared with the Con group, the OVA group mice showed a significant increase in bronchial and airway goblet cell positivity and mucus production. Compared with the OVA group, the GPH, Mixture, or DEX groups significantly reduced mucus secretion and goblet cell proliferation, respectively. Masson staining was used to detect collagen deposition and airway wall thickening. Figure 6 As shown in the figure, the positive area in the OVA group was significantly increased compared to the Con group. The positive area in the GPH, Mixture, or DEX groups was significantly decreased compared to the OVA group. These data indicate that Mixture can improve airway remodeling in allergic asthmatic mice.

[0075] 2.5 Mixture can reduce lung tissue apoptosis in mice with allergic asthma.

[0076] TUNEL staining and immunohistochemistry were used to detect whether Mixture reduced lung tissue apoptosis induced by OVA in mice with allergic asthma. The number of TUNEL-positive cells was significantly higher in the OVA group than in the Con group. Compared with the OVA group, the GPH, Mixture, or DEX groups showed a significant decrease in TUNEL-positive cells. Figure 7 In the OVA group, Bcl-2 expression was significantly lower than in the Con group, while Bax and Caspase-3 expression were significantly higher than in the Con group. However, treatment with GPH, Mixture, or DEX significantly reversed the low expression of Bcl-2 and the high expression of Bax and Caspase-3 in the lung tissue of OVA-induced allergic asthma mice. Figure 8 The results showed that Mixture reduced lung tissue apoptosis induced by OVA in mice with allergic asthma.

[0077] 2.6 Effects of Mixture on the PI3K / Akt signaling pathway in OVA-induced allergic asthma mice

[0078] The effect of mixtures on transcription factors was detected using Western blotting analysis. For example... Figure 9 As shown, the expression of PI3K, p-PI3K, and p-Akt in the OVA group was higher than that in the Con group. Conversely, the GPH, Mixture, or DEX groups significantly reduced the high expression of PI3K, p-PI3K, and p-Akt / Akt in OVA-induced allergic mice. Therefore, Mixture can alleviate lung damage in OVA-induced allergic asthmatic mice, and its mechanism of action may be related to the PI3K / Akt signaling pathway.

[0079] 3. Discussion

[0080] Allergic asthma is a heterogeneous disease caused by various triggering factors. In recent years, it has seriously threatened the health of millions and has been recognized as a global public health problem. Therefore, there is an urgent need for potentially effective treatments to control the incidence of allergic asthma.

[0081] In this study, histopathological analysis showed that OVA could induce significant pathological changes in mouse lung tissue, such as alveolar structure loss, inflammatory cell infiltration, and alveolar wall thickening

[41] . High secretion of airway epithelial mucus, goblet cell proliferation, and collagen deposition are the main markers of allergic asthma. PAS staining showed high secretion of mucus and goblet cell proliferation in the OVA group, and Masson staining also showed increased collagen deposition in the OVA group. However, the pharmaceutical composition in this application can significantly alleviate the above pathological features, revealing its protective effect against allergic asthma.

[0082] Experimental results showed that in an OVA-induced allergic asthma mouse model, the composition of this application could inhibit the production of the aforementioned cytokines such as IgE, IL-4, IL-5, IL-13 and TNF-α, indicating that the composition of this application has an anti-allergic inflammatory effect.

[0083] In summary, the results of this study suggest that the composition of this application has a protective effect against OVA-induced allergic asthma in mice, including tissue inflammation, airway hyperresponsiveness, and apoptosis. Furthermore, the composition of this application achieves good results with only a small dosage; although the dosage is higher than that of dexamethasone, it is far lower than that of *Hedyotis diffusa* extract.

[0084] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A pharmaceutical composition for treating allergic asthma, characterized in that, It is composed of the following raw materials: luteolin, arbutin, and isopyramidal lactone, with the following weight parts: luteolin 60-120 parts, arbutin 110-170 parts, and isopyramidal lactone 580-640 parts.

2. The pharmaceutical composition for treating allergic asthma according to claim 1, characterized in that, The weight proportions of each raw material are as follows: 89 parts of luteolin, 144 parts of arbutin, and 609 parts of isopyram lactone.

3. The use of the pharmaceutical composition for treating allergic asthma according to claim 1 in the preparation of a medicament for treating allergic asthma.

4. The use of the pharmaceutical composition for treating allergic asthma according to claim 1 in the preparation of a drug for treating lung inflammation caused by allergic asthma.

5. The use of the pharmaceutical composition for treating allergic asthma according to claim 1 in the preparation of a drug for reducing lung tissue apoptosis caused by allergic asthma.

6. The method for preparing the pharmaceutical composition for treating allergic asthma according to claim 1, characterized in that, The product is obtained by mixing luteolin, arbutin, and isopyram lactone.

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