A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, its preparation and application

Abstract

The present invention discloses a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, its preparation and application, and belongs to the technical field of COPD drugs. The traditional Chinese medicine composition is composed of a qi-invigorating component, a blood-activating component and a phlegm-resolving component, or a qi-invigorating component and a blood-activating component, or a qi-invigorating component and a phlegm-resolving component; wherein the qi-invigorating component is selected from any one or more combinations of ginsenoside Rg1, ginsenoside Rb1, astragaloside IV, and schisandrin A; the blood-activating component is selected from any one or more combinations of ligustrazine and ferulic acid; and the phlegm-resolving component is selected from any one or more combinations of sinigrin and quercetin. The combination of the above drugs has a significant effect on improving lung inflammatory response and inhibiting pathological remodeling of pulmonary vascular.

Description

Technical Field

[0001] The present invention belongs to the technical field of chronic obstructive pulmonary disease drugs, and in particular relates to a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, and its preparation and application. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a multifactorial, chronic inflammatory lung disease characterized by airway narrowing, emphysema, and small airway disease. These symptoms are often caused by long-term exposure to noxious irritants, particularly tobacco smoke and environmental pollutants. It is a common and frequently occurring respiratory disease, with high morbidity and mortality.

[0003] From the perspective of traditional Chinese medicine, the main pathological mechanism of COPD is deficiency of the underlying cause and excess of the superficial cause, with blood stasis, turbid phlegm, and qi deficiency as common pathogenesis. Currently, Western medicine treatments for COPD mainly focus on bronchodilators, glucocorticoids, oxygen therapy, and antibiotics to treat and alleviate symptoms. Although they can improve symptoms to a certain extent, they cannot completely reverse the course of the disease, nor can they reduce the occurrence of acute exacerbations of COPD. Long-term use may also bring more side effects, and repeated attacks of the disease are more common. In comparison, traditional Chinese medicine treatment has shown unique advantages in improving the quality of life of patients by improving the ventilation function of lung tissue and regulating the overall state of the body. However, with the development of chemical separation technology, the research on the compatibility of traditional Chinese medicine that only stays at the level of Chinese herbal medicine pieces can no longer meet the needs of the modernization of traditional Chinese medicine.

[0004] In the field of Traditional Chinese Medicine, to address the complexity of ingredients and ambiguity of action mechanisms in traditional Chinese medicine compound prescriptions, the academic community has proposed the concept of "TCM effective ingredient group." This pharmacodynamic group, as a new paradigm for the research of the substance basis of traditional Chinese medicine, not only maintains the multi-component and multi-target therapeutic characteristics of traditional Chinese medicine, but also possesses the characteristics of modern medicines such as a relatively clear chemical substance basis, significantly improved quality controllability, and gradually clarified mechanisms of action. Theoretical studies have shown that effective ingredient groups can serve as an optimized alternative form of compound Chinese medicine, providing a new path for the modernization of traditional Chinese medicine. However, there is still a lack of a compound Chinese medicine combination specifically for COPD that uses modern composition matching concepts to accurately match various effective ingredients to form a precise efficacy and controllable quality. Summary of the Invention

[0005] In response to the above problems, in a first aspect, the present invention provides a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, wherein the traditional Chinese medicine composition is composed of a qi-invigorating component, a blood-activating component, and a phlegm-resolving component, or is composed of a qi-invigorating component and a blood-activating component, or is composed of a qi-invigorating component and a phlegm-resolving component;

[0006] Wherein, the Qi-invigorating component is selected from any one or more combinations of ginsenoside Rg1, ginsenoside Rb1, astragaloside IV, and schisandrae alcohol A;

[0007] The blood-activating component is selected from any one or more combinations of ligustrazine and ferulic acid;

[0008] The expectorant component is selected from any one or more combinations of sinigrin and quercetin.

[0009] Furthermore, the Qi-invigorating ingredient is a combination of ginsenoside Rb1 and astragaloside IV;

[0010] The blood-activating ingredient is ferulic acid;

[0011] The expectorant component is sinigrin.

[0012] Furthermore, when the Chinese medicine composition includes qi-invigorating components, blood-activating components and expectorant components, the mass ratio of the raw materials in the Chinese medicine composition is ginsenoside Rb1:astragaloside IV:sinapicoside:ferulic acid=5-40:25-200:1.5-12:2.5-20.

[0013] Furthermore, when the traditional Chinese medicine composition includes qi-invigorating components, blood-activating components and expectorant components, the mass ratio of ginsenoside Rb1, astragaloside IV, sinapicin and ferulic acid in the traditional Chinese medicine composition is 20:50:4:2.5.

[0014] Furthermore, when the Chinese medicine composition includes a qi-invigorating component, a blood-activating component, and a phlegm-resolving component, the mass ratio of each raw material in the Chinese medicine composition is:

[0015] Ginsenoside Rb1: schisandra alcohol A: sinapinesin: ferulic acid = 40:12:4:5;

[0016] Ginsenoside Rb1: astragaloside IV: sinapinic acid: ferulic acid = 40:25:8:10;

[0017] Ginsenoside Rb1: astragaloside IV: schisandra alcohol A: sinapinic acid: ferulic acid = 40:200:6:2:2.5.

[0018] Furthermore, when the traditional Chinese medicine composition includes qi-invigorating components and expectorant components, the mass ratio of the raw materials in the traditional Chinese medicine composition is: ginsenoside Rb1: astragaloside IV: schisandra alcohol A: sinapicin = 40:100:3:1.

[0019] Furthermore, when the traditional Chinese medicine composition includes a qi-invigorating component and a blood-activating component, the mass ratio of the raw materials in the traditional Chinese medicine composition is: ginsenoside Rb1: astragaloside IV: schisandra alcohol A: ferulic acid = 20:200:3:10;

[0020] Ginsenoside Rb1: astragaloside IV: schisandra alcohol A: ferulic acid = 40:50:1.5:20.

[0021] In a second aspect, the present invention provides a Chinese medicine preparation containing the Chinese medicine composition for treating chronic obstructive pulmonary disease.

[0022] Furthermore, the preparation types include granules, tablets, pills, capsules or oral liquid dosage forms.

[0023] In a third aspect, the present invention proposes the use of the traditional Chinese medicine composition in the preparation of drugs for preventing and treating chronic obstructive pulmonary disease.

[0024] Beneficial effects of the present invention:

[0025] Based on the theory of effective ingredient groups in traditional Chinese medicine, this invention defines a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, including blood-activating ingredients, expectorant ingredients, and qi-tonifying ingredients. Through a multi-component synergistic mechanism, it overcomes the limitations of weak targeting and insufficient efficacy of a single ingredient. Compared with traditional compound prescriptions, it is more targeted and can significantly improve therapeutic efficacy. Based on the characteristics and theory of Xin'an medicine's "strengthening the foundation and nourishing the essence", this invention reuses lung-tonifying and qi-invigorating drugs to develop an innovative formula for treating COPD, in which ginsenoside Rb1 and astragaloside IV are derived from two medicinal materials, raw sun-dried ginseng and astragalus, respectively, and are both monarch drugs; sinapinic acid and ferulic acid are extracted from scutellaria baicalensis seeds and ligusticum chuanxiong, respectively, and are both minister drugs, forming a monarch-minister compatibility relationship under the theory of traditional Chinese medicine. Ginsenoside Rb1 and others have anti-inflammatory activity, which can reduce inflammatory cell infiltration in the airways and reduce the release of inflammatory mediators; ingredients such as astragaloside IV may improve the immune response of COPD patients by regulating immune cell activity, thereby controlling the progression of the disease; the ratio of this drug composition has been optimized, and the compound Chinese medicine prolongs the duration of therapeutic effect through the synergistic effect of multiple ingredients, reducing the need for frequent adjustments to treatment plans.

[0026] Results from cell and animal experiments demonstrate that this combination significantly improves lung inflammation and inhibits pathological pulmonary vascular remodeling, laying the experimental foundation for elucidating its synergistic mechanism. Furthermore, the natural herbs used in this drug have relatively low side effects, and by optimizing the ratio of the drug ingredients, the dosage of ingredients that may cause side effects is reduced, thereby alleviating the physiological burden on patients.

[0027] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1A The figure shows the cell survival rate after AT2 cells were treated with ginsenoside Rg1 (Ginsenoside Rg1) at different concentrations as a monomer drug in the embodiment of the present invention;

[0030] Figure 1B The figure shows the cell survival rate after AT2 cells were treated with ginsenoside Rb1 at different concentrations as a monomer drug in the embodiment of the present invention;

[0031] Figure 1C The figure shows the survival rate of AT2 cells after the cells were treated with different concentrations of astragaloside IV as a monomer drug in an embodiment of the present invention;

[0032] Figure 1D The figure shows the survival rate of AT2 cells after treating the cells with different concentrations of Schizandrin A as a monomer drug in the embodiment of the present invention;

[0033] Figure 1E The figure shows the survival rate of AT2 cells after the cells were treated with different concentrations of sinigrin as a monomer drug in the embodiment of the present invention;

[0034] Figure 1F The figure shows the survival rate of AT2 cells after treating the cells with different concentrations of quercetin as a monomer drug in the embodiment of the present invention;

[0035] Figure 1G The figure shows the survival rate of AT2 cells after treating the cells with different concentrations of Ligustrazine as a monomer drug in the embodiment of the present invention;

[0036] Figure 1H The figure shows the survival rate of AT2 cells after treating the cells with different concentrations of ferulic acid as a monomer drug in the embodiment of the present invention;

[0037] Figure 2A Graph showing the transforming growth factor-β (TGF-β) content released into the culture medium by AT2 cells in different experimental groups in the examples of the present invention;

[0038] Figure 2B A graph showing the interleukin-8 (IL-8) content released into the culture medium by AT2 cells in different experimental groups in an embodiment of the present invention is shown;

[0039] Figure 2C A graph showing the amount of tumor necrosis factor-α (TNF-α) released into the culture medium by AT2 cells in different experimental groups according to an embodiment of the present invention is shown;

[0040] Figure 2D The figure shows the content of vascular endothelial growth factor (VEGF) released into the culture medium by AT2 cells in different experimental groups of the examples of the present invention;

[0041] Figure 2E A comparison of the levels of TGF-β, IL-8, TNF-α, and VEGF released into the culture medium by AT2 cells in different experimental groups in the embodiments of the present invention is shown;

[0042] Figure 2F shows the comprehensive scoring results of different experimental groups in the embodiment of the present invention;

[0043] Figure 3A The forced expiratory volume in 0.3 seconds (FEV 0.3 ) test results;

[0044] Figure 3B shows the test results of forced vital capacity (FVC) of rats in different experimental groups according to the embodiment of the present invention;

[0045] Figure 3C The FEV values ​​of rats in different experimental groups in the present invention are shown. 0.3 / FVC ratio results;

[0046] Figure 4A Shown are the results of pathological staining of rat lung tissue in different experimental groups in the examples of the present invention;

[0047] Figure 4B The lung injury scoring results of rat lung tissues in different experimental groups in the embodiment of the present invention are shown;

[0048] Figure 5A The expression of inflammatory factor TNF-α in BALF of rats in the embodiment of the present invention is shown;

[0049] Figure 5B The expression of inflammatory factor IL-1β in BALF of rats in the embodiment of the present invention is shown;

[0050] Figure 6A The CDT of rats in the CON group to which IL-4 was added in the embodiment of the present invention is shown. +4. Flow cytometric scatter plot of lymphocytes;

[0051] Figure 6B The CDT of rats in the CON group to which IFN-γ was added in the embodiment of the present invention is shown. + 4. Flow cytometric scatter plot of lymphocytes;

[0052] Figure 6C The CDT of rats in the MOD group to which IL-4 was added in the embodiment of the present invention is shown. + 4. Flow cytometric scatter plot of lymphocytes;

[0053] Figure 6D The CDT of rats in the MOD group to which IFN-γ was added in the embodiment of the present invention is shown. + 4. Flow cytometric scatter plot of lymphocytes;

[0054] Figure 6E The CDT of rats in the XXZ group to which IL-4 was added in the embodiment of the present invention is shown. + 4. Flow cytometric scatter plot of lymphocytes;

[0055] Figure 6F The CDT of rats in the XXZ group to which IFN-γ was added in the embodiment of the present invention is shown. + 4. Flow cytometric scatter plot of lymphocytes;

[0056] Figure 7A The CD4 T cells expressing IFN-γ in the blood of rats in each group according to the embodiment of the present invention are shown. + T cell (TH1 cell) percentage results;

[0057] Figure 7B The CD4 expressing IL-4 in the blood of each group of rats in the embodiment of the present invention is shown. + T cell (TH2 cell) percentage results;

[0058] Figure 7C The results of the ratio of TH1 to TH2 cells in the blood of rats in each group in the examples of the present invention are shown. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0060] The cells, animals, drugs, and reagents involved in the examples of the present invention are as follows:

[0061] Cells: Rat alveolar type II cells were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Cat. No. FH1099).

[0062] Drugs: Ginsenoside Rg1 (batch number: 231129), Ginsenoside Rb1 (batch number: 240109), Astragaloside IV (batch number: 240120), Schisandrin A (batch number: 230108), Sinapiginosidin (Sinigrin Potassium Salt, batch number: 231203), Quercetin (batch number: 231030), Ferulic acid (batch number: 240509), and Ligustrazine (batch number: 240105) were all purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. The mass fraction of the above ingredients (HPLC) was ≥98%.

[0063] Reagents: RPMI1640 medium (Lot No. 6124357, Thermo Fisher (Suzhou) Instrument Co., Ltd.); transforming growth factor β1 enzyme-linked immunosorbent assay kit (Lot No. GR20240710, Wuhan Gene Beauty Biotechnology Co., Ltd.), tumor necrosis factor α enzyme-linked immunosorbent assay kit (Lot No. GR20240710, Wuhan Gene Beauty Biotechnology Co., Ltd.), vascular endothelial growth factor (Lot No. GR20240710, Wuhan Gene Beauty Biotechnology Co., Ltd.), tumor necrosis factor α enzyme-linked immunosorbent assay kit (Lot No. GR20240910, Wuhan Gene Beauty Biotechnology Co., Ltd.), interleukin-8 enzyme-linked immunosorbent assay kit (Lot No. GR20240710, Wuhan Gene Beauty Biotechnology Co., Ltd.), interleukin-1-β enzyme-linked immunosorbent assay kit (Lot No. GR20240910, Wuhan Gene Beauty Biotechnology Co., Ltd.); CD3-APC (Lot No. B421860, Biolegend), IFN-γ-Alexa Fluor ®647 (lot number B422865, Biolegend), IL-4-PE (lot number B412564, Biolegend), fixative (lot number B343143, Biolegend), permeabilization reagent (lot number B340086, Biolegend); CD4-FITC (lot number 2250803, BD); stimulatory agent (lot number A10722, Lianke Biotechnology).

[0064] Instrument: Animal experiment hypoxia device, Shanghai Tawang Technology Co., Ltd.; 2m 3The fumigation chamber was manufactured by Anhui University of Traditional Chinese Medicine; Hardman cigarettes (tar content: 10 mg, nicotine content: 0.8 mg); animal lung function analysis system (Beijing Bellambo Technology AniRes2005); micropipette 200 μL Eppendorf 3120000054; micropipette 1 mL Eppendorf 3120000062; microplate reader RT-6000 Raydu; rat ventilator; rat dissection operating table (Shanghai Yuyan Scientific Instrument Co., Ltd.); flow cytometer CytoFLEX; pipette KE0003087 / KA0056573; centrifuge 80-2.

[0065] Example 1

[0066] 1.1 Use CCK-8 assay to evaluate the effect of candidate Chinese medicine monomers on AT2 cell survival rate and preliminarily screen for effective monomer components;

[0067] A cell model was established using alveolar type II epithelial cells (AT2) and the effects of different drugs on cell survival were tested: AT2 cells were cultured in RPMI1640 complete medium (containing 10% fetal bovine serum) at 37°C and 5% CO2 in an incubator. 4 / mL, 100 μL per well was inoculated into a 96-well plate. After 24 hours, the cells were divided into the following groups, with 4 replicates per group:

[0068] Control group (CON): contains only culture medium without cells, used to calibrate background absorbance;

[0069] Model group (MOD): 80 μL culture medium + 20 μL cigarette smoke extract CSE (20% CSE);

[0070] Dosage group: 80 μL culture medium + 20 μL cigarette smoke extract + selected effective monomer components.

[0071] The effective monomer components to be selected include:

[0072] Lung-tonifying and Qi-invigorating monomers: ginsenoside Rb1 (5, 10, 20, 40 μg / mL), ginsenoside Rg1 (10, 20, 40, 80 μg / mL), astragaloside IV (25, 50, 100, 200 μg / mL), schisandraside A (1.5, 3, 6, 12 μg / mL);

[0073] Expectorant monomers: sinigrin (1, 2, 4, 8 μg / mL), quercetin (1.5, 3, 6, 12 μg / mL);

[0074] Blood-activating monomers: ligustrazine (12.5, 25, 50, 100 μg / mL), ferulic acid (2.5, 5, 10, 20 μg / mL).

[0075] Each candidate efficacious monomer was prepared at the specified concentrations using dimethyl sulfoxide (DMSO, concentration <0.1%) as a cosolvent. Dosage was administered according to the groupings described above. Cells were incubated for 6, 12, 24, and 48 hours following administration. CCK-8 solution was then added for an additional hour. The CCK-8 assay systematically evaluated the temporal effects of 20% cigarette smoke extract (CSE) on AT2 cells. Absorbance (OD) was measured at 450 nm using a microplate reader, and cell viability was calculated as ((treatment group or model group - control group) / (model group - control group) × 100%).

[0076] The calculation results of cell survival rate under the action of different effective monomers are as follows Figure 1A-Figure 1H As shown, Figure 1A-1D The survival results of AT2 cells after treatment with lung-tonifying and qi-invigorating Chinese medicine monomers are shown. Figure 1E-1F The survival results of AT2 cells after being treated with expectorant monomers are shown in Figure 2. Figure 1G-1H The results of AT2 cell survival after treatment with activating blood monomers are shown. Compared with the model group (12h), there was no statistical difference in cell survival rate in the 12h treatment of ginsenoside Rb1 (5, 10, 20, 40μg / mL) and ginsenoside Rg1 (10, 20, 40, 80μg / mL) concentration groups. However, when treated for 24h, compared with the model group (24h), ginsenoside Rb1 ( Figure 1B ) and ginsenoside Rg1( Figure 1A ) All concentration groups significantly increased cell survival rate ( P <0.05, ), and there was no statistical difference in the proliferation effect between the two groups. Compared with the model group (24h), the astragaloside IV treated for 24h ( Figure 1C , 25, 50, 100, 200 μg / mL) and schisandrin A ( Figure 1D , 1.5, 3, 6, 12 μg / mL) all showed significant statistical differences ( P <0.05, ).

[0077] In the expectorant monomer, 24 hours of treatment with sinigrin ( Figure 1E , 1, 2, 4, 8 μg / mL) and quercetin ( Figure 1F , 1.5, 3, 6, 12 μg / mL) at different concentrations showed statistical significance compared with the model group (24 h) ( P <0.05, ). Compared with the model group (24h), the blood-activating monomers treated with different concentrations of ligustrazine ( Figure 1G , 12.5, 25, 50, 100 μg / mL) and ferulic acid ( Figure 1H, 2.5, 5, 10, 20 μg / mL) treatment, compared with the model group, there were significant statistical differences ( P <0.05, Under these conditions, the cell survival rate of the ferulic acid-treated group was slightly higher than that of the ligustrazine-treated group.

[0078] Based on the above experimental results, ginsenoside Rb1 significantly improved cell survival at low doses, with comparable efficacy to ginsenoside Rg1. Based on the advantages of equivalence and dose economy, ginsenoside Rb1 was preferred. Similarly, ferulic acid was preferred over ligustrazine. Both sinigrin and quercetin exhibited cytoprotective effects, but sinigrin exhibited higher solubility and achieved efficacy at a lower dose, making it the preferred agent. In summary, ginsenoside Rb1, astragaloside IV, schisandrin A, sinigrin, and ferulic acid were selected as the active monomeric components.

[0079] 1.2 Based on the effective monomer components initially screened in 1.1, further screening for the best combination;

[0080] (1) Based on the CCK-8 experimental results, the concentration gradient of the effective monomer components was analyzed using the orthogonal design assistant. The orthogonal table was 5 levels and 5 factors (Table 1), generating 25 candidate component experiments (Table 2). The effective monomer components were prepared in dimethyl sulfoxide (DMSO, concentration <0.1%) as a cosolvent to form candidate component stock solutions with mass concentrations of ginsenoside Rb 120 mg / mL, ginsenoside Rg 120 mg / mL, astragaloside IV 20 mg / mL, and schisandra alcohol A 20 mg / mL; the expectorant group (Tenglizizi, Xiebai): sinapicin 20 mg / mL, quercetin 20 mg / mL; the blood circulation group (Ligusticum chuanxiong): ligustrazine 20 mg / mL, ferulic acid 20 mg / mL, and stored at -20℃ for use. The candidate component stock solutions were prepared according to the design schemes in Tables 1 and 2.

[0081] Table 1 Ratio L between groups 25 (5 6 )Orthogonal array

[0082]

[0083] Table 2 Ratio L between groups 25 (5 6 )Orthogonal design

[0084]

[0085] (2) Cell processing and detection;

[0086] Grouping and administration: AT2 cells were seeded in the same manner as in 1.1 and grouped as follows, with 3 replicates per group: AT2 cells were cultured in RPMI1640 complete medium (containing 10% fetal bovine serum) at 37°C in a 5% CO2 incubator. AT2 cells were plated at 3×10 4 / mL, 100 μL per well was seeded in a 96-well plate and grouped 24 hours later. The cells were divided into a normal group, a model group (20% CSE), and a treatment group (20% CSE + candidate agent), with triplicate wells in each group. After treatment, the cells were cultured for 24 hours. The culture supernatant was aspirated into an EP tube. ELISA was used to determine the concentrations of TNF-α, VEGF, IL-8, and TGF-β in the cell supernatant of each experimental group.

[0087] The model group (MOD) was induced by CSE to establish an in vitro model without adding Chinese medicine monomer intervention. n (n=1-25) were treated with different concentrations of Chinese medicine monomers on the basis of CSE induction, among which X1 group represented X n The concentrations of VEGF, IL-8, TNF-α and TGF-β in the cell supernatant of each group were detected by ELISA.

[0088] Control group (CON): contains only culture medium without cells, used to calibrate background absorbance;

[0089] Model group (MOD): 80 μL culture medium + 20 μL cigarette smoke extract CSE (20% CSE);

[0090] 25 dosing groups: 80 μL culture medium + 20% CSE + group 1 to group 25 (treated with different concentrations of Chinese medicine monomers).

[0091] After administration to each group, the cells were cultured for 24 hours, and the supernatant was collected into EP tubes. The concentrations of TNF-α, VEGF, IL-8, and TGF-β in the supernatant were determined by ELISA.

[0092] Based on the CCK-8 experimental results, the inter-group ratio was further optimized. Combined with the cell model activity evaluation, the comprehensive score was used to optimize the inter-group ratio of efficacy. The evaluation method is as follows:

[0093] F(VEGF)=(M / X n ) / max(M / X1:M / X 25 )×100

[0094] F(IL-8)=(M / X n ) / max(M / X1:M / X 25 )×100

[0095] F(TNF-α)=(M / X n ) / max(M / X1:M / X 25 )×100

[0096] F(TGF-β)=(M / X n ) / max(M / X1:M / X 25 )×100

[0097] Comprehensive score = [ F (VEGF)+ F (IL-8)+F(TNF-α)+F(TGF-β)] / 4

[0098] Among them, M is the model group value, X n is the intervention value of the nth medication group, max(M / X1:M / X 25 ) is M / X1 to M / X 25 The maximum value of .

[0099] Cytokine level detection results and comprehensive score results are as follows Figure 2A-2F (G1-G25 correspond to groups 1-25 respectively) and as shown in Table 3:

[0100] Table 3

[0101]

[0102] from Figure 2A It can be seen that compared with the CON group, TGF-β in the MOD group increased significantly ( P <0.05,#); Compared with the MOD group, the TGF-β level in group 1 had no statistical difference, but the TGF-β level in groups 2-25 decreased significantly ( P <0.05, There was no statistical difference between groups 2 and 3; there was no statistical difference between groups 11, 10, 6, and 4. There was no statistical difference between groups 7, 11, and 13; there was no statistical difference between groups 17, 9, 12, 8, 13, 7, and 11; there was no statistical difference between groups 14, 5, 17, 9, 12, 8, 13, and 7; there was no statistical difference between groups 15, 14, 5, 17, 9, and 12; there was no statistical difference between groups 20, 15, 14, 5, 17, and 9; there was no statistical difference between groups 16, 19, 20, 15, 14, and 5; there was no significant difference between groups 18, 20, 21, 22, 23, 24, and 25; compared with the MOD group, the TGF-β levels in groups 18, 20, 21, 22, 23, 24, and 25 decreased ( P <0.05, ), and there were statistical differences between the groups 1-17 and 19.

[0103] from Figure 2B It can be seen that compared with the CON group, the IL-8 in the MOD group increased significantly ( P <0.05,#); There was no statistical difference in IL-8 levels between groups 1 and 2, but the IL-8 levels in groups 3-25 decreased significantly ( P <0.05, There was no statistical difference between groups 2 and 3, and between groups 4 and 5; there was no statistical difference between groups 11, 6, 7, and 17; there was no statistical difference between groups 10, 8, 11, 6, and 7; there was no statistical difference between groups 12, 13, 16, 20, 25, 21, 15, and 14; there was no statistical difference between groups 24, 22, 9, 19, 12, 13, 16, 20, 25, 21, and 15; there was no statistical difference in IL-8 between groups 18 and 23; the IL-8 level decreased most significantly between groups 18 and 23 ( P <0.05, ), and there were statistical differences between the groups 1-17, 19-22, and 24-25.

[0104] from Figure 2C It can be seen that compared with the CON group, the TNF-α level in the MOD group was significantly increased ( P <0.05,#). Compared with the MOD group, the TNF-α levels in group 1 and group 5 showed no statistical difference, while the TNF-α levels in groups 2, 3, 4, and groups 6-25 decreased significantly ( P <0.05, ). There was no significant difference in TNF-α levels between groups 2, 3, 4, and 5; and no significant difference was observed between groups 6, 13, 15, and 17. Similarly, there was no statistical difference in TNF-α levels between groups 12, 11, 9, 14, 8, 13, and 17; and no significant difference was observed between groups 7, 12, 11, 9, 14, and 8. In addition, there was no statistical difference in TNF-α levels between groups 16, 10, 7, 12, 11, and 9; there was no statistical difference between groups 21 and 25; and no significant difference was observed between groups 18, 20, 22, 23, and 24. Among them, compared with the MOD group, the TNF-α levels of groups 18, 20, 22, 23, and 24 decreased ( P <0.05, ), and there were statistical differences between groups 1-17 and groups 19, 21, and 25.

[0105] from Figure 2D It can be seen that compared with the CON group, the VEGF in the MOD group increased significantly ( P<0.05,#); Compared with the MOD group, the VEGF levels in groups 1, 2, and 5 showed no statistically significant difference, while the VEGF levels in groups 3, 4, and groups 6-25 decreased significantly ( P <0.05, There was no statistical difference in VEGF levels among groups 4, 24, 17, 15, and 8; there was no statistical difference in VEGF levels among groups 19, 4, 24, 17, and 15; there was no statistical difference in VEGF levels among groups 3, 10, 9, 14, 19, and 4; there was no statistical difference in VEGF levels among groups 7, 6, 16, 3, 10, 9, 14, and 19; there was no statistical difference in VEGF levels among groups 22, 12, 7, 6, 16, and 3; there was no statistical difference in VEGF levels among groups 13, 22, 12, 7, 6, and 16; there was no statistical difference among groups 25, 13, 22, 12, and 7; there was no statistical difference among groups 23, 18, 25, 13, 22, and 12; there was no statistical difference among groups 21, 20, 23, and 18. Compared with the MOD group, the VEGF levels of groups 18, 20, 21, and 23 decreased ( P <0.05, ), and there were statistical differences between groups 1, 17, 19, 22, 24, and 25.

[0106] from Figure 2E 、 Figure 2F As can be seen from Table 3, the compatibility of each group can reduce the expression of cytokines to varying degrees. Based on the statistical analysis and market prices of each cytokine TNF-α, VEGF, IL-8, and TGF-β, group 18 was finally obtained as the best combination scheme, that is, the Chinese medicine composition was ginsenoside Rb1 20μg / mL, astragaloside IV 50μg / mL, sinapicin 4μg / mL, and ferulic acid 2.5μg / mL. The best compatibility ratio of each efficacy group was: lung-tonifying and qi-invigorating group [ginsenoside Rb1-astragaloside IV (20:50)] - phlegm-resolving group (sinapicin) - blood-activating group (ferulic acid) was 20:50:4:2.5.

[0107] Example 2

[0108] The optimal Chinese medicine composition determined in Example 1 was prepared with a ratio of 20:50:4:2.5 for the lung-tonifying and qi-invigorating group [ginsenoside Rb1-astragaloside IV (20:50)], the phlegm-resolving group (sinapidin) and the blood-activating group (ferulic acid). The specific process was as follows:

[0109] Raw material pretreatment: Weigh 10.68 mg / kg (kg corresponding to the mass of rats), astragaloside IV 26.69 mg / kg, sinapine 2.14 mg / kg, and ferulic acid 1.33 mg / kg respectively. During the weighing process, place each raw material in a clean and dry environment, control the environmental humidity ≤ 45%, and the temperature at 20 - 25 °C. Control the weighing error of each component within the range of ±0.5%.

[0110] Homogenization and mixing: Place the weighed ginsenoside Rb1, astragaloside IV, sinapine, and ferulic acid into a three-dimensional motion mixer, and add 0.5 - 2% of the total weight of microcrystalline cellulose as a glidant. Under the protection of inert gas, perform gradient mixing at a rotational speed of 15 - 20 r / min: Mix for 30 minutes in the first stage, and let it stand for 10 minutes to allow the material to settle fully; in the second stage, increase the rotational speed to 25 r / min and continue mixing for 45 minutes to ensure that each component reaches a statistically uniform distribution, and the relative standard deviation (RSD) of the mixing uniformity ≤ 2%.

[0111] Forming the preparation: Make the uniformly mixed raw materials into capsules.

[0112] It should be noted that the screened traditional Chinese medicine composition in this example can be added with pharmaceutically acceptable excipients in a certain proportion, and can also be made into drugs in different dosage forms such as granules, tablets, pills, or oral liquids according to conventional preparation methods for the treatment of COPD.

[0113] Example 3

[0114] Establishment, grouping, and administration of the COPD rat model of phlegm and blood stasis obstructing the lung: Select 40 SPF-grade SD rats aged 6 - 8 weeks with a body weight of (200 ± 20) g, purchased from the Henan Experimental Animal Center, and the animal license number is (SCXK(Yu)2020 - 0005). Approved by the Experimental Animal Welfare and Ethics Review Committee of the First Affiliated Hospital of Anhui University of Chinese Medicine (AHUCM-rats-2024161). The animals are housed in a room with a 12h / 12h day-night cycle lighting, the temperature is maintained at 22 - 24 °C, and the humidity is 50%. The animals are allowed to eat and drink freely.

[0115] The previous modeling method of the research group was continued (references: ① Li Zegeng, Wang Chuanbo, Peng Bo, Tong Jiabing, Yang Cheng, Zhang Sichun. Establishment of rat model of chronic obstructive pulmonary disease with phlegm and blood stasis obstructing lung syndrome, Tianjin Traditional Chinese Medicine, 2010, 27(1):43-45. ② Zhu Jie, Wang Baoqin, Li Zegeng, Lu Mei, Peng Qinghe, Yang Cheng, Tong Jiabing. Qibai Pingfei capsule can reduce the levels of calcineurin and nuclear factor of activated T cells c3 (NFATc3) in chronic obstructive pulmonary disease [J]. Journal of Cellular and Molecular Immunology, 2015, 31(11):1497-1501.), and a composite factor was used to establish a rat model of COPD with phlegm and blood stasis obstructing lung syndrome. According to the principle of "exhaustion leads to loss of Qi" (Suwen•Jutonglun), rats were forced to swim for 30 minutes every day in a constant temperature water tank (40±2)℃ to make them tired and consume lung Qi; then they were placed in a 1m 3 In a fumigation chamber, 20 filter-less cigarettes were smoked for 1 hour daily. Finally, the rats were placed in a constant-temperature (22°C–24°C) hypoxic plexiglass chamber, where nitrogen was purged. An automatic oxygen meter was used to regulate the oxygen concentration to (10±0.5)%, and a CO2 sensor maintained the CO2 in the chamber at 0.03% for 7 hours daily. Modeling was performed 6 days per week for 4 consecutive weeks. After one week of adaptive feeding, the rats were randomly divided into 5 groups: a normal group (CON group), a COPD model group (MOD group), a low-dose group (XD group), a medium-dose group (XZ group), and a high-dose group (XG group), with 8 rats in each group. Rats in the CON and MOD groups were gavaged with an equal volume of sterile saline (10 mL / kg); the XG group received a dose of 40.84 mg / kg, the XZ group received a dose of 20.42 mg / kg, and the XD group received a dose of 10.21 mg / kg, all once a day for two weeks, a total of 14 days. The effective group was gavaged with a suspension of ginsenoside Rb1, astragaloside IV, sinapicin, and ferulic acid determined in Example 1 in a mass ratio of 20:50:4:2.5.

[0116] 3.1. Pulmonary function test:

[0117] Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution (50 mg / kg). After being fully anesthetized, tracheotomy was performed and the rats were connected to an animal lung function analysis system. Pulmonary function indicators of the rats were detected and recorded, including forced vital capacity (FVC), forced expiratory volume in 0.3 seconds (FEV 0.3 ) and FEV 0.3 The ratio of FVC to FVC (FEV 0.3 / FVC).

[0118] The results are as follows Figure 3A 、 Figure 3B 、 Figure 3C As shown in the figure, it can be seen that compared with the CON group, the FEV0.3 / mL, (FEV 0.3 / FVC)% and FVC / mL were significantly decreased ( P <0.01,##). Compared with the MOD group, the lung function parameters in the XG group were improved ( P <0.01, ). This showed that their lung function improved significantly after drug treatment.

[0119] It should be noted that the embodiments of the present invention and the accompanying drawings 、 , #, ##, &, && are all statistically significant, and their specific explanations are as follows: Compared with the control group (#, P <0.05, ##, P <0.01); compared with the model group ( , P <0.05, , P <0.01); compared with the XZ group (&, P <0.05,&&, P <0.01). The subsequent graphs and tables are labeled in the same way.

[0120] 3.2 Hematoxylin-eosin (HE) staining:

[0121] Fixed rat lung tissue was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). HE staining was observed microscopically, and the pathological severity of peribronchial and alveolar inflammation was assessed. Peribronchial and alveolar inflammation were scored and semi-quantitatively analyzed based on the presence and intensity of inflammatory cell infiltration in the surrounding area. Inflammation around the bronchi and vessels was scored according to the following parameters: no inflammatory response (0 points); occasional inflammatory cell shedding (1 point); a thin layer of inflammatory cells (1-5 cells thick) around most bronchi and vessels (2 points); and a thick layer of inflammatory cells (>5 cells thick) around most bronchi and vessels (3 points). Alveolar inflammation was scored according to the following parameters: normal cells (0 points); a small amount of inflammatory cell infiltration (1 point); an inflammatory cell ring >1 layer of cells (2 points); an inflammatory cell ring >2-4 layers of cells (3 points); and an inflammatory cell ring >4 layers of cells (4 points).

[0122] The staining of lung tissues of rats in each group is shown in Figure 4A The lung injury score results are shown in Figure 4BAs shown, it can be seen that the alveolar structure of the rats in the normal group showed no obvious damage. The endothelial cells were arranged normally, and there were a small number of inflammatory cells in each layer of the tube wall. In the MOD group, destruction and fusion of the alveolar structure, luminal exudate, inflammatory cell infiltration of the tube wall and luminal stenosis were observed. In the low-dose group using the drug, the alveolar structure showed characteristics of destruction and fusion, exudate appeared inside the lumen, inflammatory cell infiltration existed in the tube wall, and the lumen was significantly narrowed. In the XZ group, the lung pathological condition improved, the alveolar damage was alleviated, and some inflammatory cell infiltration was seen in each layer of the tube wall. In the XG group, the lung pathological changes also showed an improving trend, the degree of alveolar damage was reduced, and a small number of inflammatory cells were still seen in each layer of the tube wall. The lung inflammation score showed that compared with CON, the MOD bronchial and perivascular scores increased ( P <0.05, #); compared with MOD, the bronchial and perivascular scores of the XG group decreased ( P <0.05, ).

[0123] 3.3. ELISA detection of BALF inflammatory factors:

[0124] Bronchoalveolar lavage fluid (BALF) was collected from rats, centrifuged, and the supernatant was collected and stored at -80°C for future use. The levels of inflammatory factors IL-1β and TNF-α in the BALF supernatant of rats in each group were detected according to the ELISA kit operating instructions.

[0125] The results are as follows Figure 5A and Figure 5B As shown in Figure 2, compared with the CON group, the levels of IL-1β and TNF-α inflammatory factors in the MOD group were significantly increased ( P <0.01, ##). Compared with the MOD group, the levels of IL-1β and TNF-α inflammatory factors in the XD group, XZ group, and XG group were significantly reduced ( P <0.01, ). This showed that the revealed fraction could effectively inhibit the inflammatory response in the COPD rat model, and its mechanism of action was closely related to reducing the levels of pro-inflammatory factors TNF-α and IL-1β.

[0126] 3.4 Flow cytometry detection of CD4 + T lymphocytes:

[0127] Take cells from CON group, MOD group and significant effect group XXZ group (corresponding to the significant effect high dose group XG group) and take 100ul (adjust the cell concentration to 1×10^ 7 / mL) was added to the flow cytometry tube. The corresponding antibodies were added and TH1 / TH2 (CD3, CD4) were incubated in the dark for 15 minutes. After adding the fixative and membrane permeabilization agent to detect TH1 and TH2 cells, appropriate amounts of IFN-γ and IL-4 were added and incubated at room temperature in the dark for 20 minutes. The cells were washed with PBS and resuspended, and detected using a flow cytometer. The flow cytometer test results of rats in different experimental groups are shown as follows. Figures 6A-6F shown.

[0128] The test results of TH1 and TH2 are as follows Figures 7A-7C As shown in the figure, it can be seen that compared with the normal group, the number of TH1 cells in the abdominal aorta blood of rats in the model group increased ( P <0.01, ##), the number of TH2 cells did not change significantly; the TH1 / TH2 ratio increased ( P <0.01, Compared with the MOD group, the number of TH1 cells in the abdominal aorta blood of rats in the XXZ group was decreased ( P <0.01, ); the number of TH2 cells did not change significantly; the TH1 / TH2 ratio decreased ( P <0.01, ). This showed that the effective group (XXZ) improved COPD inflammation by regulating the balance of TH1 / TH2.

[0129] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The mass ratio of the raw materials in the pharmaceutical composition is ginsenoside Rb1:astragaloside IV:sinapiside:ferulic acid=5-40:25-200:1.5-12:2.5-20.

2. The pharmaceutical composition for treating chronic obstructive pulmonary disease according to claim 1, characterized in that: The mass ratio of ginsenoside Rb1, astragaloside IV, sinapinic acid and ferulic acid in the pharmaceutical composition is 20:50:4:2.5; Or ginsenoside Rb1: astragaloside IV: sinapinic acid: ferulic acid = 40:25:8:

10.

3. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The mass ratio of each raw material in the pharmaceutical composition is: Ginsenoside Rb1: schisandra alcohol A: sinapinesin: ferulic acid = 40:12:4:5; Or it could be ginsenoside Rb1: astragaloside IV: schisandra alcohol A: sinapinic acid: ferulic acid = 40:200:6:2:2.

5.

4. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The mass ratio of the raw materials in the pharmaceutical composition is: ginsenoside Rb1: astragaloside IV: schisandra alcohol A: sinapicoside = 40:100:3:

1.

5. A pharmaceutical composition for treating chronic obstructive pulmonary disease, characterized in that: The mass ratio of the raw materials in the pharmaceutical composition is: ginsenoside Rb1: astragaloside IV: schisandra alcohol A: ferulic acid = 20:200:3:10; Ginsenoside Rb1: astragaloside IV: schisandra alcohol A: ferulic acid = 40:50:1.5:

20.

6. A pharmaceutical preparation, characterized in that A pharmaceutical composition for treating chronic obstructive pulmonary disease comprising the composition according to any one of claims 1 to 5.

7. The pharmaceutical preparation according to claim 6, characterized in that Preparation types include granules, tablets, pills, capsules or oral liquid dosage forms.

8. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing and treating chronic obstructive pulmonary disease.

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