A Tibetan medicine composition for moistening the lungs and resolving phlegm, its preparation method and application

Abstract

This invention discloses a Tibetan medicine composition for moistening the lungs and resolving phlegm, its preparation method, and its application, belonging to the field of pharmaceutical technology. The Tibetan medicine composition for moistening the lungs and resolving phlegm disclosed in this invention is made from the following raw materials in parts by weight: 5-20 parts of Rhododendron microphylla, 1-10 parts of Rhodiola rosea, 1-10 parts of Datura stramonium root leaves, and 1-10 parts of licorice. The preparation method of the composition of this invention includes: preparing the medicinal materials according to the proportions, pulverizing them into fine powder, sieving, mixing, adding an appropriate amount of water to form pills, drying, and polishing to obtain water pills. This invention also discloses the application of the above-mentioned Tibetan medicine composition in the preparation of drugs for treating respiratory diseases. The composition of this invention is effective and safe for use in treating respiratory diseases, especially chronic bronchitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a Tibetan medicine composition for moistening the lungs and resolving phlegm, its preparation method and application. Background Technology

[0002] Chronic bronchitis is a chronic nonspecific inflammation of the trachea, bronchial mucosa, and surrounding tissues caused by infectious or non-infectious factors. Clinical manifestations are mainly cough and sputum production, sometimes accompanied by wheezing. It typically lasts for more than two years, with a cumulative duration of more than three months per year, after excluding other diseases that could cause cough, sputum production, and wheezing.

[0003] The exact cause of chronic bronchitis is not fully understood. Common factors include infections such as viral, mycoplasma, and bacterial infections, which are important causes of the development and progression of chronic bronchitis. In addition, impaired immune function, airway hyperresponsiveness, age, and other bodily factors are also related to the occurrence and development of chronic bronchitis.

[0004] In current treatment, chronic bronchitis is primarily managed symptomatically, including cough suppressants, expectorants, and bronchodilators. When a patient has a concurrent infection, anti-infective treatment is necessary. Regarding prognosis, some patients with chronic bronchitis can achieve symptom control, while others may develop chronic obstructive pulmonary disease (COPD) or even chronic cor pulmonale. Severe cases can significantly impact daily life and work.

[0005] Therefore, there is still a clinical need for drugs that are effective and safe for treating chronic bronchitis. Summary of the Invention

[0006] The purpose of this invention is to provide a Tibetan medicine composition with lung-moistening and phlegm-reducing effects, which is effective and safe for respiratory diseases, especially chronic bronchitis.

[0007] A second objective of this invention is to provide a method for preparing the composition.

[0008] A third objective of this invention is to provide applications of the composition.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] The present invention discloses a Tibetan medicine composition for moistening the lungs and resolving phlegm, which is made from the following raw materials in parts by weight: 5-20 parts of Rhododendron simsii, 1-10 parts of Rhodiola rosea, 1-10 parts of Daphne odora root leaf, and 1-10 parts of Glycyrrhiza uralensis.

[0011] In some embodiments of the present invention, the product is made from the following raw materials in parts by weight: 8-15 parts of Rhododendron simsii, 3-8 parts of Rhododendron simsii, 3-8 parts of Alpinia officinarum leaf, and 3-8 parts of licorice.

[0012] In some embodiments of the present invention, the product is made from the following raw materials in parts by weight: 10 parts of Rhododendron simsii, 5 parts of Rhododendron simsii, 5 parts of Alpinia officinarum leaf, and 5 parts of licorice.

[0013] In some embodiments of the present invention, a pharmaceutically acceptable carrier is also included.

[0014] In some embodiments of the present invention, the Tibetan medicine composition is a preparation made from Rhododendron simsii, Rhodiola rosea, Alpinia zerumbet leaves and licorice as raw materials, plus a pharmaceutically acceptable carrier, using conventional methods.

[0015] In some embodiments of the present invention, the preparation is an oral preparation, preferably a solid oral preparation, and more preferably a pill.

[0016] The present invention discloses a method for preparing a Tibetan medicine composition for moistening the lungs and resolving phlegm, comprising the following steps:

[0017] Prepare the medicinal materials according to the proportions, grind them into fine powder, sift them, mix them evenly, add an appropriate amount of water to make pills, dry them, polish them, and you will get water pills.

[0018] Alternatively, each medicinal material can be pulverized into a fine powder, sieved, mixed in proportion, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained.

[0019] In some technical solutions of this invention, the powder is pulverized into fine powder and then passed through a No. 6 sieve.

[0020] In some embodiments of the present invention, the water pills are weighed 0.2-0.4g each, more preferably 0.3g.

[0021] The Tibetan medicine composition disclosed in this invention is used in the preparation of drugs for treating respiratory diseases.

[0022] In some embodiments of the present invention, the respiratory diseases include pneumonia, tracheitis, bronchitis, and pharyngitis; preferably chronic bronchitis.

[0023] The small-leaved rhododendron described in this invention is the Rhododendron capitulata, a plant belonging to the Ericaceae family. Rhododendron capitatum Dried flowers from Maxim.

[0024] The Rhodiola rosea described in this invention is Rhodiola grandiflora, a plant belonging to the Crassulaceae family. Rhodiola crenμlata The dried roots and rhizomes of (Hook. f.et Thoms.) H. Ohba.

[0025] The highland coriander leaf described in this invention is the dried leaf of the coriander plant, belonging to the Brassicaceae family. Coriander, known as "Niuma" in Tibetan, is a plant unique to high-altitude regions, growing at altitudes above 3500 meters.

[0026] The licorice mentioned in this invention is licorice, a plant belonging to the legume family. Glycyrrhiza uralensis Fisch., Licorice inflata Glycyrrhiza inflata Bat. or Licorice root Glycyrrhiza glabra Dried roots and rhizomes of L.

[0027] Tibetan medicine theory holds that physiological activities and the occurrence of diseases are related to imbalances in the "three factors" (Lung, Tripa, and Pekan). Lung represents energy, Tripa represents heat, and Pekan represents nutrients. A balance among these three factors ensures good health, while imbalance leads to illness.

[0028] Rhododendron simsii: In Tibetan medicine, Rhododendron simsii is believed to have the effects of clearing heat and detoxifying, relieving cough and asthma. It is mainly used for "Chiba" disease, which is a febrile disease, such as lung heat and cough. Rhododendron simsii can clear heat toxins from the body and alleviate the symptoms of febrile diseases.

[0029] Rhodiola rosea: In Tibetan medicine, Rhodiola rosea is considered a tonic and strengthening medicine, primarily used to nourish the lungs and replenish qi. It is effective in treating deficiency-related illnesses, especially lung deficiency cough and shortness of breath. Rhodiola rosea can enhance lung function and improve the body's immunity.

[0030] Highland coriander root and leaves: This medicine has the effect of relieving cough and expectoration, promoting the expulsion of phlegm and alleviating cough symptoms. In Tibetan medicine theory, it is mainly used for "Peigen" disease, which is a disease related to the imbalance of body fluid metabolism.

[0031] Licorice: In Tibetan medicine, licorice is a commonly used harmonizing agent, capable of harmonizing the properties of various medicines, enhancing their efficacy, and reducing their side effects. Licorice also has lung-moistening and cough-relieving effects, providing some relief for symptoms such as cough and excessive phlegm.

[0032] In conclusion, this Tibetan medicine formula is scientifically designed and ingeniously formulated. Rhododendron molle clears heat and detoxifies for febrile diseases; Rhodiola rosea nourishes lung qi and treats deficiency; Daphne odora root and leaves relieve cough and phlegm for fluid imbalances; and licorice harmonizes the medicinal properties and moistens the lungs to relieve cough. The four herbs work synergistically to clear heat and detoxify, moisten the lungs and replenish qi, and relieve cough and phlegm. This combination reflects the Tibetan medicine's theory of treating the root cause of disease and restoring the balance of the three factors (body, lung, and intestinal).

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention features a scientifically designed and ingeniously formulated composition. The components work synergistically to clear heat and detoxify, moisten the lungs and replenish qi, and relieve cough and phlegm. Pharmacological tests show that the composition of this invention has a good antitussive effect and a significant therapeutic effect on chronic bronchitis, inhibiting fibrosis in mice with chronic bronchitis. Mechanistically, the composition controls the activity of the NF-κB signaling pathway, controls the decrease in SOD activity and the accumulation of MDA peroxide, thereby regulating the imbalance of oxidation and antioxidation in mice and preventing oxidative damage to the respiratory organs caused by the imbalance of oxidation and antioxidation. The therapeutic effect of this composition on chronic bronchitis is dose-dependent; the absence of any one component significantly reduces the therapeutic effect. Safety tests show that the composition of this invention has good safety. Attached Figure Description

[0035] Figure 1 The graph shows the results of detecting the expression levels of inflammatory factors such as TNF-α, IL-6, and IL-1β in different groups of Experiment Example 2.

[0036] Figure 2 The image shows the results of the detection of chitinase-3-like1 and macrophage inflammatory protein-2 (MIP-2) levels in different groups of Experiment Example 2.

[0037] Figure 3 The graph shows the results of detecting the expression levels of anti-inflammatory factors IL-4 and IL-10 in different groups of the chronic bronchitis mouse model of Experiment Example 2;

[0038] Figure 4 Images of HE, Sirius red, and Masson staining of lung tissue from different groups of mice in Experiment Example 2;

[0039] Figure 5 The images show CD3, CD4, and CD8 staining patterns of lung tissue from different groups of mice in Experiment Example 2, indicating inflammation grading.

[0040] Figure 6 The staining images of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) in lung tissues of mice from different groups in Experiment Example 2 are shown.

[0041] Figure 7 Staining images of NF-κB signaling pathway proteins B-raf and MyD88 in lung tissues of mice from different groups in Experiment Example 2;

[0042] Figure 8 The diagram shows the safety test results for Example 3.

[0043] In the figure, Control represents the normal control group, Model represents the model control group, Low represents the low-dose group of the composition, Mid represents the medium-dose group of the composition, and High represents the high-dose group of the composition. RP - The group with one missing ingredient in the composition is indicated by DM, which represents the positive control group. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for illustrative purposes only and not for limiting the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0045] Unless otherwise specified, all quantities mentioned in this application refer to parts by weight.

[0046] Example 1

[0047] This embodiment discloses a method for preparing the composition of the present invention. The raw materials for the composition in this embodiment are:

[0048] 10 parts of Rhododendron simsii, 5 parts of Rhododendron simsii, 5 parts of Alpine root leaves, and 5 parts of licorice.

[0049] The preparation method is as follows: the above four ingredients are pulverized into fine powder, passed through a No. 6 sieve, mixed evenly, and then a suitable amount of water is added to form pills. The pills are dried and polished to obtain water pills with a specification of 0.3g / pill.

[0050] Example 2

[0051] This embodiment discloses a method for preparing the composition of the present invention. The raw materials for the composition in this embodiment are:

[0052] 20 parts of Rhododendron simsii, 10 parts of Rhododendron simsii, 8 parts of Alpine root leaves, and 8 parts of licorice.

[0053] The preparation method is as follows: the above four ingredients are pulverized into fine powder, passed through a No. 6 sieve, mixed evenly, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained with a specification of 0.4g / pill.

[0054] Example 3

[0055] This embodiment discloses a method for preparing the composition of the present invention. The raw materials for the composition in this embodiment are:

[0056] Five parts of Rhododendron simsii, three parts of Rhododendron simsii, one part of Alpine root leaves, and one part of licorice.

[0057] The preparation method is as follows: the above four ingredients are pulverized into fine powder, passed through a No. 6 sieve, mixed evenly, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained with a specification of 0.2g / pill.

[0058] Example 4

[0059] This embodiment discloses a method for preparing the composition of the present invention. The raw materials for the composition in this embodiment are:

[0060] 15 parts of Rhododendron simsii, 8 parts of Rhododendron simsii, 10 parts of Alpine root leaves, and 10 parts of licorice.

[0061] The preparation method is as follows: the above four ingredients are pulverized into fine powder, passed through a No. 6 sieve, mixed evenly, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained with a specification of 0.25g / pill.

[0062] Example 5

[0063] This embodiment discloses a method for preparing the composition of the present invention. The raw materials for the composition in this embodiment are:

[0064] 8 parts of Rhododendron simsii flowers, 1 part of Rhododendron simsii, 3 parts of Alpine root leaves, and 3 parts of licorice.

[0065] The preparation method is as follows: the above four ingredients are pulverized into fine powder, passed through a No. 6 sieve, mixed evenly, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained with a specification of 0.35g / pill.

[0066] Experimental Examples 1-3

[0067] Test Examples 1 and 2 disclose pharmacodynamic studies of the compositions of the present invention, and Test Example 3 discloses safety studies of the compositions of the present invention. Specifically, Test Example 1 used an ammonia nebulization inhalation method to establish a mouse model of chronic bronchitis (CB) for pharmacodynamic studies; Test Example 2 used an intratracheal injection of LPS to establish a mouse model of chronic bronchitis (CB) for pharmacodynamic studies.

[0068] The experimental animals used in Experimental Examples 1-3 of this invention are:

[0069] Healthy C57BL mice, 6-8 weeks old and weighing 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. The animals were housed in a specific pathogen-free (SPF) environment with 12 hours of light / 12 hours of darkness, a temperature controlled at 20-26℃ with a temperature difference of no less than 3℃, and humidity at 40%-70%. They had free access to water and food and underwent acclimatization for one week before animal experiments began.

[0070] The drugs, reagents, and consumables used in Experiment Examples 1-3 of this invention:

[0071] The composition described in this experimental example was prepared according to the method of Example 1.

[0072] The composition described in this experiment is missing one ingredient, which is a drug prepared according to the method of Example 1 that does not contain Rhododendron simsii.

[0073] Dextromethorphan Hydrobromide (DM).

[0074] The Luninex assay kit was purchased from R&D Company, and the antibodies are listed in Table 1.

[0075] Table 1 Immunohistochemical antibodies used in this study

[0076]

[0077] Experimental Example 1

[0078] This experimental example discloses the establishment of a chronic bronchitis (CB) mouse model using ammonia water nebulization inhalation method, and examines the cough suppression rate of the composition of the present invention on CB mice to evaluate its antitussive effect.

[0079] 1. Experimental Methods

[0080] Seventy C57BL / 6J mice weighing 18-22g were randomly divided into seven groups, with half males and half females. These groups were: normal control group, model control group (saline), positive drug control group (20mg dextromethorphan / kg), high-dose combination group (6g / kg), medium-dose combination group (3g / kg), low-dose combination group (1.5g / kg), and combination group lacking one ingredient (6g / kg).

[0081] Except for the normal control group, all other groups were administered the corresponding drugs once daily by gavage, while the model control group received physiological saline, for 7 consecutive days. One hour after the last administration, 0.2 mL of 25 wt% concentrated ammonia solution was added to a 20 mL beaker, which was immediately covered with an inverted 1000 mL beaker. Mice were placed in the beaker and stimulated for 20 seconds. Coughing was observed within 5 minutes, the number of coughs was recorded, and the cough suppression rate was calculated. The cough suppression rate was calculated using the following formula:

[0082] Cough suppression rate % = (1 - number of coughs in the dosage group / number of coughs in the blank control group) × 100%.

[0083] 2. Data Analysis

[0084] Experimental data are expressed as mean ± standard deviation (x±s), and statistical analysis was performed using SPSS 16.0 statistical software. One-way ANOVA was used to analyze the variance between different groups, with P<0.05 considered statistically significant and P<0.01 considered statistically significant.

[0085] 3. Results:

[0086] The cough suppression rate of the composition of the present invention in a mouse model of chronic bronchitis (CB) induced by ammonia nebulization inhalation is shown in Table 2.

[0087] Table 2. Cough suppression rate of the compositions of the present invention (n=10, x±s)

[0088]

[0089] Note: Data in the table are mean ± standard error. Compared with the model control group, * P <0.05,** P <0.01, *** P <0.001.

[0090] As shown in Table 2, the high-dose group of the composition effectively inhibited coughing in mice (p<0.001). The medium-dose and low-dose groups of the composition also had an inhibitory effect on coughing in mice (p<0.001), and the inhibition rate of coughing in mice decreased sequentially with decreasing dosage. The group lacking one ingredient still had an inhibitory effect on coughing in mice (p<0.05), but its effect was significantly reduced, and its effect was basically the same as that of the low-dose group.

[0091] Experimental Example 2

[0092] This experimental example discloses a mouse model of chronic bronchitis (CB) established by intratracheal injection of LPS to investigate the pharmacodynamics of the composition of the present invention.

[0093] This experiment evaluated the effects of the composition of this invention on the level of inflammatory cells and pathological changes in a mouse model of chronic bronchitis and the mice with chronic obstructive pulmonary disease (COPD) by observing the general condition of the mice and the pathological morphology of the lung tissue (gross observation, HE, Sirius red, and Masson pathological sections). The levels of pro-inflammatory factors TNF-α, IL-6, and IL-1beta, anti-inflammatory factors IL-2, IL-4, and IL-10, and macrophage inflammatory proteins MIP-2 and chitinase 3-like1 in the serum of the model mice were detected using a liquid chromatography-mass spectrometry (Luminex) chip. The mechanism of action of the composition of this invention in anti-inflammatory and mucin-inhibiting effects on the model mice was explored. The expression of CD3, CD4, and CD8 in the lung tissue of the model mice was detected using immunohistochemistry, and the effects of the composition of this invention on the expression of inflammatory cells in the lung tissue were compared and analyzed. The expression levels of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) in lung tissue were detected to study the extent to which the composition of this invention has antioxidant free radical and lipid peroxidation effects on mice, to evaluate the therapeutic effect of the composition of this invention on chronic bronchopneumonia in mice, to detect the expression levels of NF-κB signaling pathway proteins B-raf and MyD88, to explore the effect of the composition of this invention on the activation of the NF-κB signaling pathway in mouse lung tissue, and to study the mechanism of the composition of this invention on the regulation of airway inflammation and mucus secretion.

[0094] 1. Experimental Methods

[0095] Seventy healthy male C57BL mice were randomly divided into 7 groups:

[0096] The groups were divided into a normal control group, a model control group (physiological saline), a positive drug control group (20 mg dextromethorphan / kg), a high-dose group of the composition (6 g / kg), a medium-dose group of the composition (3 g / kg), a low-dose group of the composition (1.5 g / kg), and a group of the composition lacking one ingredient (6 g / kg), with 10 animals in each group.

[0097] A mouse model of chronic bronchitis was established using intratracheal injection of LPS. ① Anesthesia: Mice were anesthetized for subsequent procedures. ② Tracheal exposure: Mice were fixed supine on the operating table, exposing the trachea. ③ LPS injection: LPS was injected intratracheally. An intravenous cannula was used instead of a endotracheal tube, quickly inserted into the trachea, and LPS (200 μg / 200 μL) was injected. -1④ Repeat the procedure: Repeat the above procedure on day 7 / 14. ⑤ Suture the wound: After feeding continuously for 1 week, suture the wound. ⑥ Administer medication: Except for the normal control group, the other groups were given the corresponding drugs once a day by gavage, and the model control group was given physiological saline for 7 consecutive days. ⑦ Detection: After completing the above work, cytokine detection and pathological section examination were performed.

[0098] 2. Cytokine detection

[0099] 1) Blood was collected from mouse eyeballs to separate serum;

[0100] 2) Remove the cytokine kit from the refrigerator and allow it to reach room temperature.

[0101] 3) Dissolve the standard in the kit according to the instructions, let stand for 10 minutes, then dilute the standard 4-fold and set aside for use.

[0102] 4) Premix the beads with ultrasound for 30 seconds, then vortex for 5 minutes to fully disperse the beads.

[0103] 5) After mixing, add the beads evenly to the well plate at a rate of 50 μl / well, place the well plate on a magnetic rack, and shake off the liquid.

[0104] 6) According to the information of the preset well plate, add 50 μl of standard or sample to the well plate, seal the plate and place it on a shaking table at 600-800 rpm / min at room temperature for 2 hours.

[0105] 7) Wash the plate twice with 200μl / well washbuffer.

[0106] 8) Add 50 μl of secondary antibody per well, seal the plate, and place it on a shaking table at 600-800 rpm / min for 1 h at room temperature.

[0107] 9) Wash the plate twice with 200μl / well washbuffer.

[0108] 10) Add 50 μl / well of PE, seal the plate, and place it on a shaking table at 600-800 rpm / min at room temperature for 30 min.

[0109] 11) Wash the plate twice with 200 μl / well washbuffer.

[0110] 12) Add 120 μl of reading solution per well, seal the plate, and place it on a shaking table at 600-800 rpm / min for 5 min at room temperature.

[0111] 13) Read the values ​​on a Luminex 200 machine.

[0112] 14) Calculation results using the 5p-log method.

[0113] 3. Pathological examination

[0114] 3.1 Embedding and Paraffin Sectioning Preparation

[0115] 1) Tissue fixation: After collection, lung tissue samples were quickly placed in 4% neutral formalin solution and fixed on a shaker for 6-8 hours. The fixed tissue was then rinsed in running water for 30-60 minutes, and finally rinsed again with triple-distilled water to remove any residual formalin fixative.

[0116] 2) Tissue dehydration: The tissues were placed in 75%, 85%, and 95% graded alcohol solutions for 1 hour each, and then in anhydrous ethanol-1 and anhydrous ethanol-2 solutions for 30 minutes each, to carry out graded dehydration.

[0117] 3) Clearing: Immerse the tissue in xylene-1 and xylene-2 ​​solutions for 30 min.

[0118] 4) Paraffin infiltration: The tissue was immersed in liquid paraffin-1 and liquid paraffin-2 at 60℃ for 1 hour.

[0119] 5) Embedding: After embedding the tissue using an automatic embedding machine, place it in a -20℃ refrigerator for later use.

[0120] 3.2 H&E staining

[0121] 1) Sectioning: Take the frozen paraffin tissue from the -20℃ freezer, make 4µm serial sections, and then bake the sections in a 70℃ oven for 6 hours.

[0122] 2) Dewaxing: The sections were dewaxed and hydrated according to the standard procedure: xylene solution 10min×3, anhydrous ethanol 3min×2, 95% alcohol, 85% alcohol, 75% alcohol each 3min, rinsed with running water for 5min, and washed with distilled water for 3min×3 times.

[0123] 3) Hematoxylin staining: Stain with hematoxylin solution for 1 min, then rinse with running water for 30 s.

[0124] 4) Color separation: 1% hydrochloric acid ethanol for 3 seconds, running water for 10 minutes to return to blue, and distilled water for 1 minute to wash.

[0125] 5) Eosin staining: Stain with 0.5% eosin solution for 10 seconds, then rinse with distilled water for 2 seconds.

[0126] 6) Dehydration: 75% alcohol, 95% alcohol A, 95% alcohol B, anhydrous ethanol A, and anhydrous ethanol B for 2 seconds each, xylene A and xylene B solutions for 2 minutes each.

[0127] 7) Mounting: Mount the slides with neutral resin, observe the staining effect under an optical microscope, and acquire images using the DP Controller image acquisition system.

[0128] 3.3 Immunohistochemical staining

[0129] 1) Dewaxing: Place the baked slices in xylene A and xylene B solutions for 10 min each, then in anhydrous ethanol A, anhydrous ethanol B, 95% ethanol, 85% ethanol, and 75% ethanol for 3 min each, rinse with running water for 5 min, and rinse with distilled water 3 times, 3 min each time.

[0130] 2) Peroxidase blocking: Add 3% hydrogen peroxide to the tissue section to block endogenous peroxidase and incubate at room temperature for 10 min. Rinse with distilled water and place in PBS for 10 min.

[0131] 3) Antigen retrieval: Place the slides in Tris-EDTA buffer at pH 8.0 and incubate at 95°C for 40 min, then allow to cool naturally to room temperature. Rinse with PBS for 10 min.

[0132] 4) Immunoassay: Add a certain concentration of the corresponding primary antibody, approximately 50 μl per slide, using PBS as a blank control. Incubate at 37°C for 45 min. Rinse with PBS for 10 min. Then add a certain concentration of secondary antibody, approximately 50 μl per slide, and incubate at 37°C for 45 min. Rinse with PBS for 10 min.

[0133] 5) DAB color development: Add the DAB substrate solution and observe the color development under a microscope. Terminate the reaction when appropriate. Rinse with distilled water.

[0134] 6) Hematoxylin counterstaining: Immerse the sections in hematoxylin staining solution for 2 minutes, then rinse with distilled water for 30 seconds.

[0135] 7) Dehydration and clearing: Place the slices in 80% ethanol, 95% ethanol-1, 95% ethanol-2, anhydrous ethanol A, and anhydrous ethanol B solutions for 10 seconds each, and xylene A and xylene B solutions for 2 minutes each.

[0136] 8) Mounting: Mount the slide with neutral quick-drying adhesive, observe under an optical microscope, and acquire images using the DP Controller image acquisition system.

[0137] 4. Data Analysis

[0138] Experimental data are expressed as mean ± standard deviation (x±s), and statistical analysis was performed using SPSS 16.0 statistical software. One-way ANOVA was used to analyze the variance between different groups, with P<0.05 considered statistically significant and P<0.01 considered statistically significant.

[0139] 5. Results:

[0140] 5.1 Macroeconomic Indicators

[0141] The model animals exhibited symptoms such as coughing, sneezing, runny nose, rapid breathing, rales in the respiratory tract, nose scratching, huddling together, curling up and becoming lethargic, squinting, abnormal mental state, sluggishness, decreased appetite, weight loss or slow weight gain, and dull, yellowish fur. After treatment with the high-dose composition, the mice showed significant reduction in coughing, sneezing, runny nose, and rapid breathing, and their mental state returned to normal, their responsiveness increased, and their appetite returned to normal. Treatment with the medium-dose and low-dose groups of the composition reduced bronchitis symptoms in the mice, but the therapeutic effect was dose-dependent. The group lacking one ingredient in the composition still showed a therapeutic effect, but the effect was lower than that of the medium-dose group.

[0142] 5.2 Morphological observation of lung tissue

[0143] Macroscopic observation revealed that the model group animals exhibited enlarged lung volume, minimal collapse upon thoracotomy, dull color, blunted edges, soft texture, and reduced elasticity compared to normal lung films. Both lungs showed varying degrees of congestion, and the bronchi were red, swollen, and petechial. After treatment with a high dose of the composition, dissection of the mice revealed normal lung volume, pinkish color, smooth edges, soft and elastic texture, and no congestion. Treatment with the medium-dose and low-dose groups showed some relief of symptoms compared to the model group, but the therapeutic effect was significantly worse than that of the high-dose group. The group lacking one ingredient still showed a therapeutic effect, but its efficacy was significantly weaker than that of the high-dose group.

[0144] 5.3 Peripheral blood serum immune marker tests

[0145] Compared with the model group, the expression levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were significantly decreased in the high-dose and medium-dose groups of the composition (p<0.001). The low-dose group of the composition showed significantly lower levels of inflammatory factors compared to the model group (p<0.05). The group lacking one ingredient in the composition also effectively controlled the secretion of inflammatory factors in the peripheral blood of mice (p<0.05). Figure 1 As shown.

[0146] Compared with the model group, the levels of chitinase-3-like1 and macrophage inflammatory protein-2 (MIP-2) in lung tissue were significantly decreased in the high-dose and medium-dose groups of the composition (p<0.001). The inflammatory factors in the low-dose group were significantly lower than those in the model group (p<0.05). The group lacking one ingredient in the composition also showed control of peripheral blood inflammatory factor secretion in mice (p<0.05). Figure 2 As shown. Compared with the model control group, the levels of the anti-inflammatory cytokines interleukin-10 (IL-10) and interleukin-4 (IL-4) in the peripheral blood of mice in the high-dose and medium-dose groups of the composition were significantly increased (p<0.001). The anti-inflammatory factors in the low-dose group of the composition were significantly higher than those in the model control group (p<0.05). The group lacking one ingredient in the composition also promoted the secretion of anti-inflammatory factors in the peripheral blood of mice (p<0.05). Figure 3 As shown.

[0147] 5.4 Pathological examination of lung tissue

[0148] After resection and light microscopy, numerous inflammatory cells (monocytes, lymphocytes, plasma cells) infiltrated the lung tissue, trachea, and bronchial mucosa of the model control mice, with a small number of neutrophils. Alveolar dilation and alveolar cavity rupture were observed, with exfoliated cells within the lumen. Alveoli ruptured and largely fused, and squamous metaplasia and emphysema were observed in the tracheal epithelial cells. Bronchial mucosal epithelial cells were swollen, with goblet cells and glandular hyperplasia, ciliated cell degeneration and shedding, and abundant inflammatory exudate filling the lumen. Significant lesions were observed in the small bronchi, bronchioles, and terminal bronchi, with stenosis or obstruction.

[0149] HE analysis of the lungs of mice in the high-dose group of the composition showed smooth tracheal and bronchial mucosa with little inflammatory cell infiltration, minimal alveolar dilation, no alveolar rupture, a small amount of cell shedding within the lumen, normal trachea, and no emphysema. No obvious lesions were observed in the bronchi, bronchioles, bronchioles, and terminal bronchi.

[0150] The symptoms of mice in the medium-dose group and the low-dose group of the composition were alleviated when their lungs were dissected and compared with those in the model group, but the treatment effect was significantly worse than that in the high-dose group of the composition.

[0151] The group with one missing ingredient still has a therapeutic effect, but its therapeutic effect is significantly worse than that of the high-dose group of the composition.

[0152] Masson's fibrosis analysis and Sirius red staining showed that: mice in the model control group exhibited significant fibrin hyperplasia; mice in the high-dose group showed no obvious fibrin in their lungs; mice in the medium-dose and low-dose groups showed a small amount of fibrosis in their lungs upon dissection; and mice in the group lacking one ingredient still showed significant fibrin in their lungs, but to a lesser degree than the model control group. Figure 4 As shown.

[0153] After staining with CD3, CD4, and CD8, a large number of inflammatory cells infiltrated the lung parenchyma and trachea of ​​mice in the model control group. A small number of inflammatory cells appeared in the lungs of mice in the high-dose group of the composition. In the medium-dose and low-dose groups of the composition, inflammatory cell infiltration was still present in the lungs, but significantly reduced compared to the model control group. The group lacking one ingredient in the composition could still control inflammation in mice, but the therapeutic effect was significantly lower than that of the high-dose group. Figure 5 As shown.

[0154] Immunohistochemical staining showed that, compared with the model control group, the expression levels of myeloperoxidase (MPO) and malondialdehyde (MDA) were significantly reduced in the high-dose group of the composition. The medium-dose group and the low-dose group of the composition also inhibited the secretion of MPO and MDA in the lungs of mice. The group lacking one ingredient in the composition had an inhibitory effect on the secretion of MPO and MDA in mice, but it was significantly weaker than that in the high-dose group of the composition.

[0155] Compared with the model control group, the high-dose treatment group of the composition showed significantly increased activities of superoxide dismutase (SOD) and reduced glutathione (GSH) (p<0.001). The medium-dose and low-dose groups of the composition still effectively promoted the expression of SOD and GSH. The group lacking one ingredient in the composition could still effectively counteract the lung damage caused by oxygen free radicals in mice, but the therapeutic effect was significantly lower than that of the high-dose group (e.g., ...). Figure 6 ).

[0156] Immunohistochemical staining showed that, compared with the model control group, the expression levels of NF-κB signaling pathway proteins B-raf and MyD88 were significantly reduced in the high-dose group of the composition. The medium-dose and low-dose groups of the composition also inhibited the expression of B-raf and MyD88 in mouse lungs. The group lacking one ingredient in the composition showed an inhibitory effect on the secretion of B-raf and MyD88 in mice, but the effect was significantly weaker than that in the high-dose group (e.g., ...). Figure 7 ).

[0157] Experimental Example 3

[0158] This test example discloses a safety evaluation test of the composition of the present invention.

[0159] Healthy mice were given a high dose of the composition of the present invention once a day for 7 consecutive days by gavage. After taking samples from major organs such as lungs, liver, spleen, kidneys, stomach, and intestines, HE slides were prepared to observe the damage of the composition of the present invention to the major organs of mice and to evaluate its safety.

[0160] The results showed that after continuous administration of high-dose drugs to mice for 7 days, no lesions were observed in the HE morphology of the major organs of the mice, indicating that the composition of the present invention has good safety. Figure 8 As shown.

[0161] In summary, the composition of the present invention has a significant therapeutic effect on inhibiting ammonia-induced cough in mice, and this effect is dose-dependent.

[0162] The composition of this invention has a significant therapeutic effect on LPS-induced chronic bronchitis in mice. It can control the inflammatory response of LPS-induced chronic bronchitis in mice and inhibit fibrosis. Mechanistically, the composition controls the activity of the NF-κB signaling pathway, reduces SOD activity, and increases the accumulation of MDA peroxide, thereby regulating the imbalance between oxidation and antioxidation in mice and preventing oxidative damage to the respiratory organs caused by this imbalance. The therapeutic effect of the composition on chronic bronchitis is dose-dependent; the absence of any one ingredient significantly reduces the therapeutic effect. Safety tests show that the composition has good safety.

[0163] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention used to illustrate the technical solutions of the present invention, and are not intended to limit the invention, nor are they intended to limit the scope of the patent. Any modifications or refinements made to the main design concept and spirit of the present invention that are not of substantial significance, but which still solve the same technical problem as the present invention, should be included within the scope of protection of the present invention. In addition, the direct or indirect application of the technical solutions of the present invention to other related technical fields are similarly included within the scope of patent protection of the present invention.

Claims

1. A Tibetan medicine composition for treating chronic bronchitis, characterized in that, It is made from the following raw materials in parts by weight: 5-20 parts of Rhododendron simsii, 1-10 parts of Rhododendron simsii, 1-10 parts of Alpine Rhizome Leaf, and 1-10 parts of Licorice. The small-leaved rhododendron mentioned is *Rhododendron capitulata*, a plant in the Ericaceae family. Rhododendron capitatum Dried flowers from Maxim.

2. The Tibetan medicine composition for treating chronic bronchitis according to claim 1, characterized in that, It is made from the following raw materials in parts by weight: 8-15 parts of Rhododendron simsii, 3-8 parts of Rhodiola rosea, 3-8 parts of Alpine rhizome leaves, and 3-8 parts of licorice.

3. The Tibetan medicine composition for treating chronic bronchitis according to claim 1, characterized in that, It is made from the following ingredients in parts by weight: 10 parts Rhododendron molle, 5 parts Rhodiola rosea, 5 parts Alpine ginseng root leaves, and 5 parts licorice.

4. A Tibetan medicine composition for treating chronic bronchitis according to any one of claims 1-3, characterized in that, It also includes pharmaceutically acceptable carriers.

5. The Tibetan medicine composition for treating chronic bronchitis according to claim 4, characterized in that, The Tibetan medicine composition is a preparation made from Rhododendron simsii, Rhodiola rosea, Alpine root leaves and licorice as raw materials, plus a pharmaceutically acceptable carrier, using conventional methods.

6. The Tibetan medicine composition for treating chronic bronchitis according to claim 5, characterized in that, The preparation is an oral preparation.

7. The Tibetan medicine composition for treating chronic bronchitis according to claim 5, characterized in that, The preparation is a solid oral dosage form.

8. The Tibetan medicine composition for treating chronic bronchitis according to claim 5, characterized in that, The preparation is in the form of pills.

9. A method for preparing a Tibetan medicine composition for treating chronic bronchitis according to any one of claims 1-8, characterized in that, Includes the following steps: Prepare the medicinal materials according to the proportions, grind them into fine powder, sift them, mix them evenly, add an appropriate amount of water to make pills, dry them, polish them, and you will get water pills. Alternatively, each medicinal material can be pulverized into a fine powder, sieved, mixed in proportion, and then made into pills with an appropriate amount of water. After drying and polishing, water pills are obtained.

10. A method for preparing a Tibetan medicine composition for treating chronic bronchitis according to claim 9, characterized in that, The specifications for the water pills are that each pill weighs 0.2-0.4g.

11. A method for preparing a Tibetan medicine composition for treating chronic bronchitis according to claim 10, characterized in that, The water pills are 0.3g in size.

12. The Tibetan medicine composition according to claims 1-8 is used in the preparation of a medicament for treating chronic bronchitis.

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