Application of Sweeping mahonia lung-heat-clearing cough-relieving composition in treatment of chronic obstructive pulmonary disease

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to novel application of a Sweeping Tianhu lung-clearing and cough-relieving composition in treatment of chronic obstructive pulmonary disease. Animal experiment studies show that the Sweetio lung-clearing and cough-relieving composition can significantly improve the lung function of COPD mice and inhibit bronchial abnormal remodeling. The action mechanism of the composition is related to inhibition of lung tissue cell apoptosis, alleviation of inflammatory cell infiltration and improvement of abnormal remodeling of bronchus, which indicates that the Sweeping Litu composition for clearing away lung-heat and relieving cough has obvious pharmacological activity of resisting COPD (chronic obstructive pulmonary disease).

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to the novel use of the Saori Lao lung-clearing and cough-relieving composition in the treatment of chronic obstructive pulmonary disease. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a destructive lung disease characterized by incompletely reversible airflow limitation. This airflow limitation typically progresses and is associated with an abnormal inflammatory response of the lungs to harmful particles or gases. Acute exacerbations and complications affect the severity of the disease and individual prognosis. Although COPD is an airway disease, its systemic effects on the whole body should not be ignored. The main symptoms include chronic cough, sputum production, shortness of breath or dyspnea, wheezing, and chest tightness. Incompletely reversible airflow limitation is a prerequisite for the diagnosis of COPD. An FEV1 / FVC ratio <70% and an FEV1 <80% of predicted value after inhalation of bronchodilators confirm incompletely reversible airflow limitation. A small number of patients may not have cough or sputum production symptoms, but only have an FEV1 / FVC ratio <70% and an FEV1 ≥80% of predicted value on pulmonary function tests; in such cases, after ruling out other diseases, COPD can also be diagnosed.

[0003] Saorilao Lung-Clearing and Cough-Relieving Combination is derived from the Mongolian medicine formula Saorilao-7 Decoction Powder. As a major formula in Mongolian medicine for treating lung heat diseases, it has been used for thousands of years and has significant efficacy.

[0004] This formula consists of North American ginseng, Terminalia chebula, Melia toosendan, Rubia cordifolia, Gardenia jasminoides, Lithospermum erythrorhizon, and Lithospermum erythrorhizon. Mongolian medicine theory holds that North American ginseng has the effects of clearing lung heat, relieving cough, and moistening the lungs; it is supplemented with Rubia cordifolia, Lithospermum erythrorhizon, and Lithospermum erythrorhizon, which clear blood heat, to enhance the effect of clearing lung heat, reflecting the unique understanding of the efficacy of North American ginseng and the special characteristics of its formulation in Mongolian medicine.

[0005] Given the symptoms of chronic obstructive pulmonary disease (COPD), traditional Chinese medicine should actively participate in the prevention and treatment of COPD and develop symptomatic prescriptions. This invention uses the Saorilao lung-clearing and cough-relieving composition to study its antagonistic effects in order to find methods for the prevention and treatment of COPD and to explore the wider application of Saorilao lung-clearing and cough-relieving capsules. Summary of the Invention

[0006] This invention provides a novel use of the Saori Lao lung-clearing and cough-relieving composition in the treatment of chronic obstructive pulmonary disease. The Saori Lao lung-clearing and cough-relieving composition of this invention comprises: 550-650 parts of North American ginseng, 80-120 parts of Terminalia chebula, 50-80 parts of Melia toosendan, 80-120 parts of Gardenia jasminoides, 80-120 parts of Lithospermum erythrorhizon, and 80-120 parts of Rubia cordifolia.

[0007] Preferably, the lung-clearing and cough-relieving composition comprises: 600 parts of North American ginseng, 100 parts of Terminalia chebula, 60 parts of Melia toosendan, 100 parts of Gardenia jasminoides, 100 parts of Lithospermum erythrorhizon, 100 parts of Lithospermum erythrorhizon, and 100 parts of Rubia cordifolia.

[0008] The preparation form of the lung-clearing and cough-relieving composition of the present invention is decoction, capsule, granule, tablet or pill.

[0009] Preferably, the formulation of the lung-clearing and cough-relieving composition is a capsule.

[0010] The above seven medicinal materials are extracted, concentrated, dried, pulverized, granulated, and encapsulated to obtain the final product. Each capsule contains 0.4 grams.

[0011] The dosage of the Saorilao Qingfei Zhike Capsules is 3 capsules each time, 3 times a day.

[0012] Saorilao Lung-Clearing and Cough-Relieving Capsules have the effects of clearing lung heat, relieving cough, and eliminating phlegm. Clinically, they are used to treat acute exacerbations of chronic bronchitis. Inventively, this invention uses Saorilao Lung-Clearing and Cough-Relieving Capsules for research on the prevention and treatment of COPD. Animal studies have shown that administration of Saorilao Qingfei Zhike Capsules at 400 mg / kg and 800 mg / kg significantly improved lung function indicators in COPD mice, including significantly downregulating inspiratory capacity (IC, ml); downregulating static compliance (Cst, ml / cmH2O) as measured by pulmonary function instruments; significantly downregulating lung index (mg / g); improving airway wall thickness in COPD mice; significantly inhibiting inflammatory cell infiltration; significantly reducing collagen deposition in lung tissue of COPD-induced model mice; significantly inhibiting excessive mucus secretion in bronchial walls of COPD-induced model mice; inhibiting COPD-induced apoptosis; significantly downregulating pro-inflammatory cytokines TGF-β1, IL-1β, TNF-α, and IL-6 in bronchoalveolar lavage fluid and increasing the inflammatory suppressor cytokine IL-10; and significantly downregulating disease marker genes (Pro-Col I, Pro-Col...) in COPD-induced model mice. It can reduce the expression levels of TIMP-1 and TIMP-2; inhibit the phosphorylation activation of key protein molecules in the MAPK signaling pathway; significantly reduce the protein level of α-SMA, with effects comparable to or superior to the positive control drug NAC, and has significant lung protective function.

[0013] The Sorrel Lung-Clearing and Cough-Relieving Capsules of this invention can significantly improve lung function in COPD mice and inhibit abnormal bronchial remodeling. Its mechanism of action is related to inhibiting lung tissue cell apoptosis, reducing inflammatory cell infiltration, and improving abnormal bronchial remodeling, suggesting that Sorrel has significant anti-COPD pharmacological activity. This invention expands the disease areas for the application of Sorrel. Attached Figure Description

[0014] Figure 1 Effects of Saori Lao Qingfei Zhike Capsules on Respiratory Function and Lung Index in COPD Model Mice

[0015] A. Inspiratory capacity (IC) measured by pulmonary function testing; B. Static compliance (Cst) measured by pulmonary function testing; C. Lung index. Results are expressed as mean standard error (SE), n≥6. #, p<0.05 compared with normal group; ##, p<0.01 compared with normal group; *, p<0.05 compared with untreated model group; **, p<0.01 compared with untreated model group.

[0016] Figure 2 Effects of Saorilao Qingfei Zhike Capsules on Abnormal Airway Remodeling and Inflammatory Infiltration in COPD Model Mice

[0017] A, Typical images of H&E staining (200); B, Statistical results of airway wall thickness measurement; C, Pathological scoring of lung tissue after H&E staining. Results are expressed as mean (mean) and standard error (SE), n≥6. ##, p<0.01 compared with the normal group; *, p<0.05 compared with the untreated model group; **, p<0.01 compared with the untreated model group.

[0018] Figure 3 Effects of Saorilao Qingfei Zhike Capsules on Collagen Deposition in Lung Tissue of COPD Model Mice

[0019] A, Typical image of Masson staining (200); B, Relative collagen area in mouse lung tissue obtained by Image-pro plus 5.1 analysis. Results are expressed as mean (mean) standard error (SE) (n≥6). ##, p<0.01 compared with normal group; **, p<0.01 compared with untreated model group.

[0020] Figure 4 Effects of Saorilao Qingfei Zhike Capsules on Bronchial Mucus Secretion in COPD Model Mice

[0021] A, Typical image of AB-PAS staining (400); B, Statistical results of bronchial mucus layer thickness measurement. Results are expressed as mean (mean) and standard error (SE), n≥6. ##, p<0.01 compared with the normal group; **, p<0.01 compared with the untreated model group.

[0022] Figure 5 Effects of Saorilao Qingfei Zhike Capsules on Apoptosis of Lung Tissue Cells in COPD Model Mice

[0023] A, TUNEL staining representative image; B, lung tissue cell apoptosis rate measured by TUNEL assay. Results are expressed as mean (mean) and standard error (SE) (n=10). ##, p<0.01 compared with normal group; **, p<0.01 compared with untreated model group; $$, p<0.01 compared with NAC treatment group.

[0024] Figure 6 Effects of Saorilao Qingfei Zhike Capsules on the Number of Immune Cells and Levels of Inflammatory Factors in BALF of COPD Model Mice. A, Statistical results of inflammatory cell count in BALF; B, Statistical results of neutrophil count in BALF; C, Statistical results of lymphocyte count in BALF; D, Expression level of TGF-β1 in BALF; E, Expression level of TGF-β1 in BALF; F, Expression level of IL-1β in BALF; G, Expression level of TNF-α in BALF; G, Expression level of IL-6 in BALF; H, Expression level of IL-10 in BALF; Results are expressed as mean (mean) standard error (SE) (n≥5). ##, p<0.01 compared with normal group; *, p<0.05 compared with untreated model group; **, p<0.01 compared with untreated model group.

[0025] Figure 7 Effects of Saorilao Qingfei Zhike Capsules on the Expression of Lung Fibrosis-Related Genes in COPD Model Mice

[0026] A. Expression level of Pro-Col I in lung tissue; B. Expression level of Pro-Col III in lung tissue; C. Expression level of TIMP-1 in lung tissue; D. Expression level of TIMP-2 in lung tissue; Results are expressed as mean (mean) standard error (SE) (n=3). #, p<0.05 compared with normal group; ##, p<0.01 compared with normal group; *, p<0.05 compared with untreated model group; **, p<0.01 compared with untreated model group.

[0027] Figure 8 Effects of Saorilao Lung-Clearing and Cough-Relieving Capsules on Key Proteins of Lung Tissue Inflammation and Fibrosis in COPD Model Mice Detailed Implementation

[0028] The following examples further illustrate the invention, but are not intended to limit the invention in any way.

[0029] Unless otherwise specified, all experiments in the following experiments were conducted under standard conditions or conditions recommended by the manufacturer. Active pharmaceutical ingredients (APIs) or excipients, as well as reagents or instruments whose manufacturers are not specified, are all commercially available products. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention.

[0031] Pharmacodynamic Evaluation of Saorilao Qingfei Zhike Capsule Against Chronic Obstructive Pulmonary Disease in Example 1

[0032] 1. Experimental Materials

[0033] 1.1 Experimental animals: SPF-grade C57BL / 6 mice at 10 weeks of age, weighing 18 - 22 g, a total of 60 mice. Provided by Spf(Beijing) Biotechnology Co., Ltd., production license number: SCXK(Beijing) 2019 - 0010, animal quality certificate number: 110324221104950621. All animals were housed in the Experimental Animal Center of the Institute of Materia Medica, Chinese Academy of Medical Sciences, at constant temperature and humidity, with free access to food and water.

[0034] 1.2 Modeling reagents

[0035] Lipopolysaccharide (LPS), purchased from sigma-aldrich, specification: 10 mg, product number: L2630

[0036] 1.3 Therapeutic reagents

[0037] Saorilao Qingfei Zhike Capsule is produced by Inner Mongolia Tianqi Zhongmeng Pharmaceutical Co., Ltd. Batch number: 22112009.

[0038] The positive drug acetylcysteine tablets (NAC) were purchased from Beijing Gaoji Baikang Pharmacy, specification: 0.2 g, batch number: 1002217.

[0039] 1.4 Other reagents

[0040] The Giemsa staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. Kit product number: G1015.

[0041] The TUNEL kit was purchased from Beyotime Biotechnology Co., Ltd., product number: C1088.

[0042] Paraformaldehyde was purchased from Sinopharm Chemical Reagent Co., Ltd., product number: 80096618.

[0043] Normal saline was purchased from Shijiazhuang No.4 Pharmaceutical Co., Ltd., product number:国药准H13023200.

[0044] The Smad3 antibody was purchased from Cell Signaling Technology, product number: 9523.

[0045] p-Smad3 antibody was purchased from Cell Signaling Technology, catalog number: 9520.

[0046] p38 antibody was purchased from Cell Signaling Technology, catalog number: 8690.

[0047] p-p38 antibody was purchased from Cell Signaling Technology, catalog number: 4511.

[0048] The JNK antibody was purchased from Cell Signaling Technology, catalog number: 9252.

[0049] p-JNK antibody was purchased from Cell Signaling Technology, catalog number: 9255.

[0050] The α-SMA antibody was purchased from Boster Biologics Inc., catalog number: BM0002.

[0051] 1.5 Experimental Apparatus

[0052] Tissue homogenizer, Olympus imaging system (DP72), small animal pulmonary function instrument (FV-FXM2), Biotek ELISA reader (Synergy H1), electrophoresis apparatus (DYY-7C), gel imaging system (ImageQuant LAS 4000mini).

[0053] 2. Model preparation and dosing regimen

[0054] COPD modeling scheme and experimental group settings

[0055] C57BL / 6 mice were intratracheally infused with 200 μl (1 g / L) of LPS on days 0 and 14. From day 1 to day 84, the mice were placed in a smoke chamber and passively inhaled cigarette smoke (3R4F standard cigarettes) for 4 hours each time, 6 days a week, ensuring a particulate concentration of 200 mg / m³ in the smoke chamber. 3The experiment was conducted for 12 consecutive weeks. During each fumigation session, 9 cigarettes should be lit within 30 minutes, ensuring complete combustion. Normal control mice received an equal volume of physiological saline via intratracheal instillation. Model mice were randomly divided into 5 groups based on body weight. From day 57 to 84 after modeling, the mice were administered Saori Lao Qingfei Zhike capsules at 400 mg / kg and 800 mg / kg via gavage, respectively, and acetylcysteine ​​tablets (NAC) at 78 mg / kg (human dosage calculated at 0.6 g / 70 kg body weight, mouse dosage calculated at 20 g body weight). The experiment ended on day 85, lung function was assessed, and bronchoalveolar lavage fluid (BALF) was collected. Animals were euthanized by anesthesia with an overdose of tribromoethanol, and lung tissue was collected for organ index determination and subsequent pathological and fibrosis evaluation. The normal control group and model group were administered distilled water via gavage at the same time (dose volume 10 ml / kg).

[0056] 3. Testing Plan

[0057] Lung function assessment includes inspiratory capacity (IC), static compliance (Cst), and organ index.

[0058] Tissue collection and histopathological analysis included: H&E staining to determine airway wall morphology and inflammatory infiltration; Masson staining to determine lung fibrosis; Alcian blue-periodic acid-Schiff (AB-PAS) staining to determine airway mucus secretion; TUNEL staining to determine the degree of cell apoptosis; ELISA to detect the levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β1 in mouse bronchoalveolar lavage fluid (BALF); RT-PCR; and agarose gel electrophoresis (EB staining) to detect the expression of fibrosis-related factors (ProCol I, ProCol III, and TIMP1 / 2, etc.) in lung tissue. Western blot analysis was used to detect the expression levels of fibrosis-related proteins such as α-SMA, Smad3, p-p38, p38, p-JNK, JNK, and GAPDH.

[0059] 4. Experimental Results

[0060] 4.1 Saorilao Qingfei Zhike Capsules restored respiratory function in COPD model mice and effectively reduced lung organ indices.

[0061] Experimental results showed that the IC value of mice in the model group was significantly increased, and the Cst value was also significantly elevated, indicating that the mice exhibited typical COPD pathological features. Two doses of serotonin treatment significantly improved lung function in mice, showing a significant difference compared to the model group. Figure 1 A, B). The lung index of mice in the model group was significantly increased, and treatment with two doses of Saori Lao Qingfei Zhike capsules significantly reduced the lung index. Figure 1C) suggests that it has corresponding anti-COPD pharmacological activity.

[0062] 4.2 Saori Lao Qingfei Zhike Capsules significantly reduced airway wall thickness and improved inflammatory infiltration in lung tissue in COPD model mice.

[0063] H&E staining was performed on pathological sections of lung tissue. Figure 2 A) The airway wall thickness of the lung tissue in each group of mice was statistically analyzed, and the results are shown in Table 1 and... Figure 2 As shown in the figure. Experimental results showed that, compared to the normal control group, the bronchial wall thickness of the model group mice increased by 2.7 times, exhibiting severe abnormal tissue remodeling accompanied by extensive inflammatory cell infiltration. Administration of Saori Lao Qingfei Zhike capsules could improve the occurrence of abnormal airway remodeling in COPD to varying degrees, and showed a certain dose-dependent relationship. Figure 2 B). Pathological grading results showed that the positive control drug NAC and two doses of serotonin significantly inhibited inflammatory cell infiltration and reversed lung tissue damage and pulmonary edema in COPD mice. Figure 2 C).

[0064] Table 1. Statistical analysis of the effects of serotonin and NAC on bronchial wall thickness in COPD model mice.

[0065]

[0066] 4.3 Saori Laoqingfei Zhike Capsules inhibit collagen deposition in the lung tissue of COPD model mice.

[0067] Masson staining was used to detect collagen deposition in tissues. The results showed that the normal group had relatively less collagen area in lung tissue, while the blue positive areas in lung tissue sections from COPD model mice were significantly increased, with the percentage of collagen area increasing to 15.96 times the normal value. Both doses of Saori Lao Qingfei Zhike capsules significantly reduced COPD-induced collagen deposition in the lung tissue of model mice in a dose-dependent manner. The 800 mg / kg dose group was comparable in effect to the positive control drug NAC. Figure 3 ).

[0068] 4.4 Saorilao Qingfei Zhike Capsules inhibit excessive bronchial mucus secretion in COPD model mice

[0069] After staining lung tissue pathological sections with Alcian blue-periodic acid-Schiff (AB-PAS), Figure 4 A) The results showed that the thickness of the bronchial mucus layer in the lungs of the model group mice was significantly increased, and two doses of Saori Lao Qingfei Zhike capsules could significantly inhibit excessive mucus secretion in the bronchial walls of COPD model mice, and the high-dose group had a comparable effect to the positive control drug. Figure 4 B).

[0070] 4.5 Saori Laoqingfei Zhike Capsules reduce apoptosis in lung tissue cells of COPD model mice.

[0071] The apoptosis of lung tissue cells induced by COPD was detected using deoxyribonucleotide terminal transferase-mediated nick-labeling (TUNEL). The results showed an increase in TUNEL-positive cells in the lung tissue of COPD model mice (positive cells exhibited red fluorescence, with blue fluorescence in the nucleus). The positive control drug NAC and two doses of serotonin inhibited COPD-induced apoptosis, with the higher dose of serotonin showing superior inhibition of lung tissue cell apoptosis compared to NAC. Figure 5 ).

[0072] 4.6 Saorilao Qingfei Zhike Capsules reduced the number of inflammatory cells and the level of inflammatory factors in the bronchoalveolar lavage fluid of COPD model mice.

[0073] BALF from the lungs of mice in each group was collected and cell smears were prepared. Giemsa staining was used to statistically analyze the immune cells in the BALF. The results were consistent with H&E staining: Saori Lao Qingfei Zhike capsules significantly downregulated the number of inflammatory cells in lung tissue. Figure 6 A). Staining, classification, and counting of inflammatory cells showed that the infiltration of neutrophils and lymphocytes in COPD model mice was significantly reduced after administration of two doses of serotonin. Figure 6 B, C), accompanied by the pro-inflammatory cytokine TGF-β1 ( Figure 6 D), IL-1β Figure 6 E), TNF-α Figure 6 F), IL-6 Figure 6 Downregulation of G) and the inflammatory suppressor cytokine IL-10 ( Figure 6 The increase in H) suggests that Sauris has significant pharmacological activity in resisting COPD-induced lung tissue inflammation.

[0074] 4.7 Saorilao Qingfei Zhike Capsules inhibit the expression of genes related to pulmonary fibrosis in COPD model mice.

[0075] Total RNA was extracted from lung tissue, and Pro-ColI was detected using quantitative real-time PCR. Figure 7 A), Pro-ColIII Figure 7 B) TIMP-1 Figure 7 C) and TIMP-2 Figure 7The relative expression level of D). The experimental results showed that Saorilao Qingfei Zhike Capsules could significantly downregulate the expression level of fibrosis-related genes in COPD model mice. Combined with the previous detection results of lung tissue cell apoptosis, it is suggested that Saorilao Qingfei Zhike Capsules have unique pharmacological activity in inhibiting lung damage and reversing abnormal bronchial remodeling.

[0076] 4.8 Saori Laoqingfei Zhike Capsules inhibit the expression of key proteins in lung tissue inflammation and fibrosis in COPD model mice.

[0077] Western blotting results showed that ( Figure 8 Phosphorylation levels of Smad3, P38, and JUN were low in the lung tissue of normal mice, but significantly increased after COPD modeling, suggesting activation of the MAPK signaling pathway. Saori Lao Qingfei Zhike Capsules inhibited the phosphorylation activation of key protein molecules in the MAPK signaling pathway and significantly reduced the protein level of α-SMA. These results suggest at the protein molecular level that Saori Lao can alter the inflammatory state of lung tissue in COPD model mice and has a significant lung-protective function.

Claims

1. Use of the Saori Lao Qingfei Zhike Composition in the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.

2. The use as described in claim 1, characterized in that, The lung-clearing and cough-relieving composition of the Sao Ri Lao contains: 550-650 parts of North American ginseng, 80-120 parts of Terminalia chebula, 50-80 parts of Melia toosendan, 80-120 parts of Gardenia jasminoides, 80-120 parts of Lithospermum erythrorhizon, 80-120 parts of Lithospermum erythrorhizon, and 80-120 parts of Rubia cordifolia.

3. The use as described in claim 2, characterized in that, The lung-clearing and cough-relieving composition of the Sao Ri Lao contains: 600 parts of North American ginseng, 100 parts of Terminalia chebula, 60 parts of Melia toosendan, 100 parts of Gardenia jasminoides, 100 parts of Lithospermum erythrorhizon, 100 parts of Lithospermum erythrorhizon, and 100 parts of Rubia cordifolia.

4. The use according to claim 3, characterized in that, The preparation form of the Sao Ri Lao Qing Fei Zhi Ke Composition is decoction, capsule, granule, tablet or pill.

5. The use according to claim 4, characterized in that, The formulation is in capsule form.

6. The use according to any one of claims 1-5, characterized in that, The aforementioned lung-clearing and cough-relieving composition reduces inflammatory cell infiltration and / or improves abnormal bronchial remodeling.

Images