CN116726086A

CN116726086A - Tibetan medicine composition for treating chronic obstructive pneumonia and preparation method and application thereof

Info

Publication number: CN116726086A
Application number: CN202310329708.0A
Authority: CN
Inventors: 马立明; 汪豪; 秦鹏飞; 丰玉林
Original assignee: Diqing Tibetan Autonomous Prefecture Tibetan Hospital
Current assignee: Diqing Tibetan Autonomous Prefecture Tibetan Hospital
Priority date: 2023-03-30
Filing date: 2023-03-30
Publication date: 2023-09-12

Abstract

The application discloses a Tibetan medicine composition for treating chronic obstructive pneumonia and a preparation method and application thereof. The Tibetan medicine composition is obtained by repeatedly optimizing the medicine composition and content through clinical and basic research, and is prepared from seven medicinal materials of gentian flower, ash bark, common ainsliaea herb, nutmeg, liquorice, limestuary and tamarind paste. The Tibetan medicine composition can reduce the expression level of inflammatory factors in serum and lung tissues of a mouse which is a drug effect model of chronic obstructive pneumonia, improve the lung function index, reduce the airway inflammation by activating AMPK/mTOR (mammalian induced plasma display panel/mammalian target of rotation) pathway-induced autophagy, and reduce the expression of inflammatory proteins by inhibiting MAPK pathway protein activation. The medicine is clinically suitable for treating chronic obstructive pulmonary disease, is safe and effective, has controllable quality, has better curative effect than chemical medicines such as glucocorticoid and the like which are already marketed, and is a national medicine variety which is needed in clinical urgent need.

Description

Tibetan medicine composition for treating chronic obstructive pneumonia and preparation method and application thereof

Technical Field

The application belongs to the technical field of Tibetan medicines, and in particular relates to a Tibetan medicine composition for treating chronic obstructive pneumonia as well as a preparation method and application thereof.

Background

Chronic obstructive pulmonary disease (Chronic Obstructive Pulmonary Disease, COPD, simply slow obstructive pulmonary), a disease state characterized by an incompletely reversible airflow limitation that is a recurrent, progressive progression associated with abnormal inflammatory responses of the lungs to noxious gases or noxious particles; the exact cause of COPD is not known, and is similar to emphysema. Clinically, COPD is diagnosed when chronic bronchitis or (and) emphysema patients have airflow limitation in their lung function examination and are not fully reversible. Chronic bronchitis refers to chronic, nonspecific inflammation of the bronchial wall; emphysema refers to the abnormal and durable expansion of the distal air lumen of the bronchioles at the end of the lung, accompanied by destruction of the alveolar walls and bronchioles. In addition, COPD is a wasting to the body, which can impair immunity, making patients susceptible to infectious diseases. The serious patients cause complications of other organs, such as pulmonary heart disease, pulmonary encephalopathy and the like, and the heart and the brain are seriously damaged, so that the physical health and the quality of life of people are seriously threatened. With the development of society, the serious atmospheric pollution, the increase of haze environment and the increase of smoking and other pulmonary diseases lead to the rising trend of the incidence of COPD year by year, and no effective therapeutic drug patients can not be prevented and treated in time at present, so that the disease control rate is low, the patient fatality rate is high, and the disease is one of diseases which endanger the health of people in society.

COPD has complex pathogenesis, is related to chronic inflammation, oxidative stress, protease/antiprotease unbalance and immune unbalance, clinically presents symptoms such as chronic cough, expectoration, shortness of breath, dyspnea and the like, and the conventional treatment mainly comprises cough, phlegm elimination and anti-inflammatory, and adopts a bronchodilator as main measures, and when the illness is serious, the disease is often combined with glucocorticoid to be matched with low-concentration oxygen inhalation; the bronchodilator can better relieve COPD symptoms, but has no definite anti-inflammatory effect, and can treat symptoms without root cause; although the glucocorticoid has better anti-inflammatory effect, the long-term use has great toxic and side effects, and the body can generate glucocorticoid resistance, so that the illness state is more serious. The traditional Chinese medicine has abundant experience accumulation in the aspect of pulmonary disease treatment, and has important scientific significance in searching COPD (chronic obstructive pulmonary disease) treatment medicines from traditional Chinese medicine and ethnic medicine classical formula.

Disclosure of Invention

The application aims to search a proper medicine for treating COPD from a Tibetan medicine classical prescription for performing optimization of cutting, develop a new clinical indication for treating COPD by a traditional Tibetan medicine, develop a proper medicinal formulation, and achieve the purposes of definite curative effect, increased patient compliance and safe medicine taking.

In order to achieve the above purpose, the Tibetan medicine composition provided by the application is prepared by the following scheme:

the Tibetan medicine composition for treating the chronic obstructive pneumonia is prepared from the following raw materials in parts by weight: 120-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of common knotweed herb, 19-25 parts of nutmeg, 50-60 parts of limestone, 20-30 parts of slag tamarind paste and 75-85 parts of liquorice.

Preferably, the composition is prepared from the following raw materials in parts by weight: 140-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of common knotweed herb, 19-23 parts of nutmeg, 50-55 parts of limestone, 26-30 parts of slag tamarind paste and 75-79 parts of liquorice.

The composition is added with pharmaceutically acceptable auxiliary materials to prepare a Tibetan medicine compound preparation. The preparation can be one of granules, tablets and capsules.

The preparation method of the Tibetan medicine composition for treating the chronic obstructive pulmonary disease comprises the following steps: taking gentian flower, ash bark, common lantain herb, nutmeg and liquorice according to a certain proportion, soaking the gentian flower, ash bark, common lantain herb, nutmeg and liquorice in water, decocting or heating and refluxing for extraction, filtering, concentrating filtrate to obtain concentrated extract, adding slag tamarind paste, mixing, precipitating with ethanol, concentrating supernatant, mixing with limestone, drying, crushing and sieving to obtain extract powder.

The soaking time is 30-60min, preferably 30-40min.

The above decoction or heat reflux extraction is carried out for 2-3 times, each time for 1-2 hr, and water consumption for each time is 8-14 times of the weight of the medicinal materials, preferably 8-12 times of the weight.

The ethanol precipitation is carried out by adding 95% ethanol with 3-4 times volume, and the time of the ethanol precipitation is 24 hours.

The Tibetan medicine compound provided by the application can obviously improve the chronic obstructive pulmonary disease symptoms, and the Tibetan medicine composition has obvious curative effect when being applied to preparing medicines for treating chronic obstructive pneumonia.

The specific efficacy of the drug components used in the application is as follows:

gentian flower: the product is dry flowering aerial part of Gentian veitchiorum Hemsl. The identification basis is as follows: meets the regulations of the 98 th page of the double-flower gentian item of the 2004 edition of the Tibetan autonomous area Tibetan medicine Standard. In the book of Jingzhu Ben Cao, it is recorded in Tibetan medicine that gentian flower is used as "bang Zhi Yu temperature keeping" for treating toxic diseases, various heat diseases and laryngitis heat-retention.

Cortex Fraxini Radicis: the product is dried branch skin or dry bark of Fraxinus chinensis Fraxinus Rhynchosia Hance of Oleaceae, fraxinus schinsensisensRoxb, fraxinus spinosa Lingelsh, or Fraxinus mandshurica Lingelsh. Meets the regulations of the Chinese pharmacopoeia in the year 2020 edition, part 282, page and under the ash bark item. The main treatment of the cortex Fraxini is recorded in China herbal medicine to clear heat and dry dampness; clearing liver and improving vision; relieving cough and asthma. Diarrhea due to damp-heat; leucorrhea, conjunctival congestion and swelling and pain; sore and nebula can be generated in eyes; cough and dyspnea due to lung heat.

All-grass of Lagotis: the product is dried whole plant of Lagotis japonica Maxim or Lagotis integera W.Smith. The Chinese plant Zhi records that the common lantana herb has the effects of clearing heat, reducing internal heat, diminishing inflammation and detoxifying for treating lung abscess, cough and adverse reaction, phthisis, chest fullness, purulent blood, blood heat suppurative cavity, yellow water disease and other diseases.

Nutmeg: the product is dried kernel of Myristica fragrans Houtt. The Chinese pharmacopoeia records that the nutmeg has the main function of warming the middle energizer and promoting the circulation of qi, astringing intestines and stopping diarrhea. Can be used for treating deficiency-cold in spleen and stomach, chronic diarrhea, abdominal distention and pain, anorexia, and emesis.

Licorice root: the product is dry root and rhizome of Glycyrrhiza glabra of Leguminosae. The Chinese pharmacopoeia records that the licorice has the functions of invigorating spleen, replenishing qi, clearing away heat and toxic material, eliminating phlegm, relieving cough, relieving spasm and pain, and harmonizing the medicines. Can be used for treating weakness of spleen and stomach, listlessness, debilitation, palpitation, short breath, cough with excessive phlegm, abdominal pain, limb spasm, carbuncle, swelling, sore and toxic materials, and relieving drug toxicity and intensity.

Lime bloom: the product is carbonate mineral, and contains mainly calcium carbonate (CaCO 3). The calx is recorded in the Chinese herbal medicine assembly, has the efficacy of clearing heat and tonifying lung, and is suitable for treating various lung heat diseases.

Slag tame cream: the product is a concentrated extract prepared by decocting and concentrating the residue tamarind with water. The Tibetan medicine works on four medical classics, and the slag tame paste can treat all heat symptoms, and has obvious curative effects of clearing stomach heat, clearing liver heat, clearing kidney heat and the like.

In classical "four-stage" of Tibetan medicine, it is indicated that the superior living body is formed by five elements (earth, water, fire, wind and air). The lung belongs to the bacon area, five elements are earth and water, belongs to cold, and has the functions of regulating water solution and secreting mucus. Because of the cold-dampness and the property of dispersing on the skin of the body surface and orifices, the respiratory tract is easy to be infected by external factors, thereby causing cough, thick phlegm, fever and inflammation. The disturbance of bacon causes bacon pathogen, which causes the heat, sharp, light and greasy properties of red barks to be changed to cause red barks, and causes heat syndrome, blast and various inflammations, and chronic bronchitis can be caused by long-term unhealed or repeated attacks. When five-element substances in a human body are clinically diagnosed to be deregulated, medicines with different properties and effects are used for treating the five-element substances, and cold and cool medicines are generally used for treating the five-element substances. The prescription for treating respiratory diseases, which is well recorded in the four medical classics, is a plurality of prescriptions, such as fifteen gentian pills, nine-ingredient limhua powder, three-ingredient gentian pills, six-ingredient clove pills and the like, and is clinically applicable to treating bronchitis, senile asthma and other diseases, has complex medicine flavor and has no clinical application for treating chronic obstructive pneumonia.

The gentian flower is taken as a main medicine for reasonable formulation, the cold and cool Tibetan medicine capable of treating lung heat is added to enhance the curative effect of gentian flower, and corresponding medicines capable of preventing bacon or carina caused by the cold and cool medicine are matched, so that the aim of coordinating three factors of carina, red bar and bacon is fulfilled. The specific formula is that gentian flower is taken as a main medicine, and cortex fraxini helps the gentian flower to clear heat and remove dampness; the calx helps gentian flower to clear heat and tonify lung; the herba Lagotis and the dreg domestica paste have the effects of helping the gentian flower to clear away heat and toxic materials, wherein the herba Lagotis can prevent bacon syndrome, clear away lung-heat and stomach blood stasis and discharge pus; wen Xinyao semen Myristicae can be used for preventing symptoms of spleen and stomach deficiency-cold by warming spleen and stomach and promoting qi circulation; and liquorice is matched for harmonizing various medicines, so that the spleen is strengthened, qi is enriched, and the pain is relieved. Proved by clinical trial optimization of Tibetan medicine and modern pharmacological research, the recipe is suitable for treating chronic obstructive pneumonia.

Compared with the prior art, the application has the beneficial effects that:

1. the Tibetan medicine composition provided by the application is reasonable in compatibility, and is an empirical formula combined by the inventor based on Tibetan medicine theoretical knowledge and combined with years of clinical practice.

2. The Tibetan medicine composition has less medicine odor, simple process and is beneficial to medicine production and quality control.

3. The Tibetan medicine composition is mainly prepared from seven medicinal materials of gentian flower, ash bark, common knotweed herb, nutmeg, liquorice, limhua and slag tamarind paste, and is obtained by repeatedly optimizing the medicinal composition and the proportion through clinical and basic research. The Tibetan medicine composition can reduce the expression level of inflammatory factors in serum and lung tissues of a mouse which is a drug effect model of chronic obstructive pneumonia, improve the lung function index, reduce the airway inflammation by activating AMPK/mTOR (mammalian induced inflammatory cytokine inhibitory protein) pathway induced autophagy, and reduce the expression of inflammatory proteins by inhibiting MAPK pathway protein activation. Therefore, based on a large amount of animal experimental research data, the Tibetan medicine composition can improve the lung respiratory function, exert the effects of resisting inflammation and enhancing immunity, and has no obvious toxicity and adverse reaction. The medicine is clinically suitable for treating respiratory tract inflammatory diseases such as chronic obstructive pulmonary disease, is safe and effective, has controllable quality, has better curative effect than chemical medicines such as glucocorticoid and the like which are already marketed, and is a national medicine variety which is needed in clinical urgent need.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

FIG. 1 is a graph (x 100) showing pathological section of a lung tissue HE staining of a COPD mouse by a Tibetan medicine composition of the application;

FIG. 2 is a graph (x 200) showing pathological section of HE staining of lung tissue of a COPD mouse by using the Tibetan medicine composition of the application;

FIG. 3 is a schematic diagram showing the mechanism of the action of the Tibetan medicine composition of the application on protecting the inflammatory diseases of the respiratory tract of macrophages Raw264.7 by activating the AMPK/mTOR autophagy pathway; in the graph, (A) bar graphs of the effect of LDZK at different concentrations on Raw264.7 cell viability; (B) the amount of release of the cytokine IL-1β from each group; (C) the amount of release of the cytokines IL-6 from each group; (D) the amount of release of the cytokine TNF- α from each group; (E) raw band map of AMPK/mTOR pathway protein;

FIG. 4 is a schematic representation of the mechanism of action of the Tibetan drug composition of the present application to protect alveolar epithelial cells A549 against airway inflammation by inhibiting activation of MAPK pathway; in the graph, (A) toxicity study of LPS with different concentrations on A549 cells; (B) The LDZK with different concentrations affects the bar graph on the activity of the A549 cells; (C) Protection of LPS-induced cytotoxicity by LDZK at different time points (12, 24, 48 h); (D) Original band patterns of inflammatory proteins INOS, COX2 and MAPK pathway proteins.

Detailed description of the preferred embodiments

The application is further illustrated below in conjunction with specific examples, which are provided solely for the purpose of illustrating the application in detail and are not intended to limit the scope of the application; furthermore, it is to be understood that modifications of the application in its various equivalents may fall within the scope of the application as defined in the appended claims after reading the present disclosure.

Example 1

55g of limestuary is taken, crushed and sieved (80 meshes) for standby. Weighing 120g of gentian flower, 55g of ash bark, 55g of common knotweed herb, 23g of nutmeg and 80g of liquorice, removing impurities, cutting, placing into extraction equipment, adding 10 times of water into the raw material, soaking for 30min, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a first extract, adding 8 times of water into the raw material, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a second extract, combining the two extracts, concentrating under reduced pressure to obtain a concentrated extract, adding 26g of slag tamarind extract, uniformly mixing, adding 3 times of 95% ethanol in volume of the extract mixture, stirring uniformly, standing for 24h, concentrating supernatant, merging with limestone, drying, crushing and sieving to obtain extract powder. Adding microcrystalline cellulose and lactose, mixing, granulating with 75% ethanol, and sieving to obtain granule.

Example 2

50g of lime mud is taken, crushed and sieved (80 meshes) for standby. Weighing 160g of gentian flower, 50g of ash bark, 50g of common knotweed herb, 19g of nutmeg and 75g of liquorice, removing impurities, shearing, placing into extraction equipment, adding 12 times of water by weight of the raw material medicine, soaking for 30min, heating, decocting and extracting for 2h, filtering with a 100-mesh filter cloth to obtain a first extract, adding 10 times of water by weight of the raw material medicine, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a second extract, combining the two extracts, concentrating under reduced pressure to obtain a concentrated extract, adding 30g of slag tamarind extract, uniformly mixing, adding 3 times of 95% ethanol by volume of the extract mixture, stirring uniformly, standing for 24h, concentrating supernatant, merging with limestone, drying, crushing and sieving to obtain extract powder. Adding starch, sugar powder and dextrin, granulating by conventional wet method, mixing with magnesium stearate, and tabletting.

Example 3

52.5g of limestuary is taken, crushed and sieved (80 meshes) for standby. Weighing 140g of gentian flower, 50g of ash bark, 52.5g of common knotweed herb, 21g of nutmeg and 79g of liquorice, removing impurities, cutting, placing into extraction equipment, adding 10 times of water by weight of the raw material medicine, soaking for 30min, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a first extract, adding 8 times of water by weight of the raw material medicine, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a second extract, combining the two extracts, concentrating under reduced pressure to obtain a concentrated extract, adding 28g of slag tamarind extract, uniformly mixing, adding 3 times of 95% ethanol by volume of the extract mixture, stirring uniformly, standing for 24h, concentrating supernatant, mixing with limestone, drying, crushing and sieving to obtain extract powder, adding starch with a conventional dosage, and filling into a hollow capsule to obtain the capsule.

Example 4

52.5g of limestuary is taken, crushed and sieved (80 meshes) for standby. Weighing 140g of gentian flower, 52.5g of ash bark, 52.5g of common ainsliaea herb, 21g of nutmeg and 77g of liquorice, removing impurities, cutting, placing into extraction equipment, adding 10 times of water into the raw materials, soaking for 30min, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a first extract, adding 10 times of water into the raw materials, heating, decocting and extracting for 2h, filtering with a 100-mesh filter cloth to obtain a second extract, combining the two extracts, concentrating under reduced pressure to obtain a concentrated extract, adding 28g of slag tamarind extract, uniformly mixing, adding 4 times of 95% ethanol in volume of the extract mixture, stirring uniformly, standing for 24h, concentrating supernatant, mixing with limestone, drying, crushing and sieving to obtain extract powder, adding a concentrated sucrose aqueous solution with a conventional dosage, and preparing syrup.

Example 5

55g of limestuary is taken, crushed and sieved (80 meshes) for standby. Weighing 140g of gentian flower, 52.5g of ash bark, 52.5g of common lantana leaf, 21g of nutmeg and 77g of liquorice, removing impurities, cutting, placing into extraction equipment, adding 12 times of water into the raw materials, soaking for 30min, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a first extract, adding 10 times of water into the raw materials, heating, decocting and extracting for 1h, filtering with a 100-mesh filter cloth to obtain a second extract, combining the two extracts, concentrating under reduced pressure to obtain a concentrated extract, adding 28g of slag tamarind extract, uniformly mixing, adding 3 times of 95% ethanol into the extract mixture, uniformly stirring, standing for 24h, concentrating supernatant, mixing with limestone, drying, crushing, sieving to obtain extract powder, adding a pharmaceutically acceptable wetting agent to obtain a pill, and rounding the pill by a pill making machine to obtain pills.

The said plaster is prepared by decocting radix Angelicae sinensis with 5-10 times of water for 1-2 times, and concentrating the filtrate.

Example 6

In order to study the curative effect of the Tibetan medicine compound on chronic obstructive pulmonary disease, the application adopts a method of dripping LPS into the respiratory tract and combining with atomizing papain to establish a mouse model of chronic obstructive pulmonary disease, and takes the mouse model as a study object to develop a pharmacodynamic test. The method comprises the steps of observing the weight of a mouse and blood inflammation indexes in a modeling period, carrying out initial judgment on the feasibility of the modeling method, carrying out final judgment on the feasibility of the COPD modeling method through lung tissue pathological sections, lung respiratory functions, bronchoalveolar perfusion fluid (BALF), blood inflammation cell indexes, immune organ indexes and the like of a model group mouse and a control group mouse after the experiment is finished, and determining the treatment effect of the Tibetan medicine prescription on COPD by comparing various indexes of each medicine group and the model mouse.

The present application is illustrated by the following test examples of efficacy:

test drug (drug extract powder prepared by the method of example 2), control drug (dexamethasone), lipopolysaccharide LPS, papain Papan, 60C 57 male mice, isoflurane, 25% uratan, nebulizer, hematology analyzer, (EMMS) FM forced pulmonary function detection system, tissue refiner, lipid oxidation (MDA) detection Kit, mouse IL-6ELISA Kit, mouse TNF-alpha ELISA Kit.

6.2 grouping of animals

60C 57 mice are weighed in the last day of quarantine period for 7 days, and 9 mice are randomly selected as blank groups according to weight layering; the other mice are molded together, and death numbers are removed after molding, and the mice are randomly divided into a COPD model group, a Dexamethasone (DEX) group, a Tibetan medicine low-concentration group and a Tibetan medicine high-concentration group. 8-9 per group.

6.3 modeling and administration

Except for the normal control group, 50 μl of LPS (1 mg/ml) was instilled into the nasal cavity on day 1 of the experiment, 25 μl of LPS (1 mg/ml) was instilled into the nasal cavity on days 15 and 27, and papain (1 mg/ml) was atomized after recovering the instilled LPS for 1-3 days, 1 min/day, and the mixture was atomized every other day, and molding was performed for 4 weeks.

After the molding is finished, the medicine is administrated, the normal saline for the stomach of the model group is 10ml/kg, the DEX group is administrated with 0.2mg/kg of DEX solution for the stomach, the suspension of the extract powder of the Tibetan medicine for the stomach of the low concentration group is 102.75mg/kg, the folding crude medicine is 0.9g crude medicine/kg, the suspension of the extract powder of the Tibetan medicine for the stomach of the high concentration group is 205.5mg/kg, and the folding crude medicine is 1.8g crude medicine/kg. The stomach was co-irrigated for 4 weeks. Papain (1 mg/ml) was nebulized for 1 min/min every other day for 2 weeks prior to the dosing period. The total period of the experiment was 8 weeks.

After the 8 th week of stomach irrigation is finished, 5 groups of lung function measurement are selected, after the measurement is finished, the thoracic cavity is opened to obtain lung tissues, the left lung is fixed by 4% paraformaldehyde, the right lung is weighed and then stored at-80 ℃, and later the lung is used for measuring the kit index; in addition, 3-4 mice in each group were subjected to bronchoalveolar lavage after blood was taken, BALF solution was collected, and differential leukocyte count was determined.

6.4 Experimental criteria

(1) Weight detection

The mice were subjected to weight measurement weekly, and the activity state, food intake, etc. of the mice were observed.

(2) Blood inflammatory cell index

The eyebox of the mice was collected at weeks 4 and 8, and the white blood cell count, the neutrophil count and the lymphocyte count in the blood were analyzed by an automatic blood analyzer.

(3) Inflammatory cell index in bronchoalveolar lavage fluid BALF

The skin of the throat of the mouse is cut off, and the tissues around the trachea are separated by forceps, so that the trachea is exposed. The needle of the syringe No. 7 is cut off, and the needle point is ground down again to fix the animal in the upward position. Firstly, threading two surgical sutures between tracheoesophagus, transversely cutting T-shaped incision at thyroid position by using small scissors, inserting self-made needles into trachea, respectively fastening at the position where cannula enters trachea and the distal end of cannula, connecting self-made needles with 1ml syringe to lavage 0.5ml physiological saline, staying for 10s to slowly suck back, repeating twice, making milky foam liquid, stopping when the piston is felt to suck and have resistance, transferring to EP tube, repeating above lavage process for 3 times, combining the sucked liquid, and recovering more than 80% to obtain qualified lavage liquid. The total number of leukocytes, neutrophils, lymphocytes, eosinophils were counted using an automatic hematology analyzer.

(4) IL-6, TNF-alpha assay in lung tissue

The lower right lung leaf which is not subjected to lung perfusion is weighed about 30mg according to the weight ratio of 1:9, adding PBS according to the proportion, homogenizing the lung tissue, centrifuging at 12000r and 4 ℃ for 10 minutes, taking supernatant, and strictly detecting IL-6 and TNF-alpha according to the steps of the kit.

(5) Index of pulmonary function

Mice lung function was measured at the end of week eight. The four limbs and the head and the neck of the anesthetized mice are fixed, the neck subcutaneous tissue is separated layer by layer in a blunt manner until the exposed trachea is separated, a transverse small opening is cut at the upper section of a trachea ring, the trachea cannula is inserted and then the mice are fixed by an operation line ligation, then the mice are placed into a mouse cabin of a pulmonary function instrument, the pulmonary function instrument is connected for mechanical ventilation and measurement, the measurement result of each mouse is repeated three times, and the pulmonary function index comprises 50ms forced expiratory volume (FEV 50), forced Vital Capacity (FVC), pulmonary function residual volume FRC, maximum expiratory flow PEF and maximum expiratory middle-section flow MMEF.

(6) Pathological section of lung tissue

The fixed left lung is treated according to the steps of dewatering water for wax, embedding, slicing, dewaxing to water, hematoxylin dyeing, eosin dyeing and dewatering the water-sealed slice, and finally microscopic examination is carried out, and image acquisition and analysis are carried out under the visual field of 100 times and 200 times.

(7) Immune organ index

After bronchoalveolar lavage of mice, respiratory function of mice was measured, thymus and spleen were weighed, and organ index was measured. COPD patients have reduced immunity and defenses to respiratory pathogenic microorganisms, and thymus and spleen indicators are commonly used to assess host immunity.

6.5 experimental results

(1) Molding result

(a) General state of mice in molding period

Compared with the control group, the model mice have the advantages of reduced diet and severely reduced weight, the specific results are shown in table 1, in addition, the model mice have symptoms of wheezing, nodding respiration, accelerating deepening of respiration, abdominal muscle twitching, binding and arching back and the like, and the voluntary activity is reduced to reach the mortality rate of about 25 percent.

TABLE 1 weight gain of mice in each group during the modeling period

Note that: p <0.01, P <0.05 compared to control group

(b) Index of mouse inflammation in modeling period

Table 2 analysis of the results shows that the increase in leukocytes in the blood of the model mice was extremely remarkable, and neutrophils and lymphocytes were also remarkably increased, compared with the control group, indicating that severe inflammatory responses were generated in the mice and corresponding immune responses were elicited.

TABLE 2 inflammatory cell counts in groups of mice during the modeling period (10) ⁹ /L)

Note that: p <0.01, P <0.05 compared to control group

(2) Therapeutic results

(a) The Tibetan medicine compound of the application affects the general state of COPD mice

The weight gain rate and voluntary activity of the remaining groups were significantly reduced compared to the control group; compared with the COPD model group, the Tibetan medicine compound medicine group has high autonomous activity, good state and stable increase of the weight increase rate; the weight of the model group mice increases and decreases, which indicates that the later development of COPD has a certain influence on the weight; the dexamethasone group body weight is in a descending trend, and the activity is not high. The specific weight results are shown in Table 3.

TABLE 3 weight gain rate of mice in each group after administration

Note that: p <0.01, P <0.05 compared to control; compared to COPD group, #p <0.01, #p <0.05

(b) The Tibetan medicine compound of the application affects blood inflammatory cells of COPD mice

Compared with a model group, the leucocyte WBC and the lymphocyte Lym in blood of the Tibetan medicine compound medicine group are obviously reduced, the Tibetan medicine compound medicine group is dose-dependent, and the neutrophil index is dose-dependent reduction trend. Compared with the model group, the WBC and Lym of the DEX group are remarkably reduced, but the Neu index is remarkably increased. The specific results are shown in Table 4, so that the Tibetan medicine compound extract powder has good systemic anti-inflammatory effect and more stable drug effect than DEX.

Table 4 blood inflammatory cell number of mice in each group after administration (10 ⁹ /L)

(c) The Tibetan medicine compound of the application affects the inflammatory cell index in BALF of COPD mice

Compared with a model group, the Tibetan medicine compound medicine group alveolar lavage fluid has a tendency of reducing inflammatory leucocyte WBC, and a high-concentration medicine group has a remarkable tendency of reducing; the WBC index was significantly reduced in the DEX group compared to the model group. Compared with the modeling, the eosinophil number in the BALF of the Tibetan medicine compound medicine group and the DEX group is extremely obviously reduced; the neutrophil count showed a decreasing trend without significant differences. The specific results are shown in Table 5, and preliminary show that both the Tibetan medicine compound medicine and dexamethasone DEX have lung anti-inflammatory effect.

Table 5 inflammatory cell number in BALF of mice of each group after administration (10 ⁹ /L)

(d) The Tibetan medicine compound of the application indicates IL-6 and TNF-alpha in lung tissue of COPD mice

Compared with the model group, the Tibetan medicine compound medicine group has a reduction trend of inflammatory factor IL-6 and TNF-alpha indexes, wherein the high concentration medicine group is obviously reduced, the inflammatory factor index of the DEX group is also obviously reduced, and the specific result is shown in Table 6. The index further shows that the Tibetan medicine compound medicine has obvious anti-inflammatory effect on COPD mice and is dose dependent resistant, and the anti-inflammatory effect of the Tibetan medicine compound medicine is equivalent to that of a positive medicine.

TABLE 6 IL-6, TNF-alpha content (pg/mgprot) in lung tissue of mice of each group after administration

(e) The Tibetan medicine compound of the application affects the lung function index of COPD mice

Compared with the model group, the 50ms forced expiratory volume ((FEV 50), forced Vital Capacity (FVC), maximum expiratory middle flow (MMEF) and maximum expiratory flow (PEF) of the Tibetan medicine compound medicine group all have the tendency to increase, wherein the high-concentration medicine group has the tendency to decrease the functional residual air volume PRC, wherein the high-concentration medicine group is obviously decreased, the indexes of FEV50, MMEF, PEF and FRC are all indicated to be obviously improved when the dexamethasone DEX group is compared with the model group, but the FVC index is worse than the model group index, and the specific result is shown in table 7.

TABLE 7 Lung function index of mice in each group after administration

(f) The Tibetan medicine compound of the application affects the lung tissue pathological section of the COPD mice

In order to quantify the pathological changes of the lung tissue, the results can be more clearly and directly compared, and the index table of the pathological degree of the lung tissue can be obtained by consulting the literature, as shown in the table 8. The results of pathological sections of lung tissue are specifically shown in FIG. 1 (x 100) and FIG. 2 (x 200).

Compared with the normal group, the model group has abnormal alveolar morphology, structural damage and partial alveolar cavities mutually fused into pulmonary bullae, and the alveoli can be infiltrated by inflammatory cells; the lung interstitium is infiltrated by a large amount of inflammatory cells, and the lung interstitium is obviously thickened; the bronchial wall thickens, the lumen narrows, necrosis of the epithelial cells of the wall occurs, and cell debris and mucus appear in the lumen. Compared with the model group, the alveolar injury condition of the DXM group and the low-concentration drug group is slightly improved, but the bronchus injury condition is not obviously improved; most of alveoli of the high-concentration pharmaceutical group are normal in morphology, and the fusion of part of alveoli is visible, so that inflammatory cell infiltration and bronchus injury conditions are obviously improved.

TABLE 8 evaluation of lung tissue pathological degrees of mice in each group after administration

(g) The effect of the Tibetan medicine compound on the immune organ index of the COPD mice

Compared with the normal group, the spleen index and thymus index of the model group and the Tibetan medicine compound medicine group are reduced, which indicates that the immune regulation unbalance is caused by the COPD modeling. Compared with the model group, the spleen index and thymus index of the LDZK drug group are increased in a dose-dependent manner; the spleen index and thymus index were smaller for the dexamethasone group than for the model group, and the specific results are shown in Table 9. Thus, dexamethasone severely reduces the immunity of the organism, while LDZK lozenge extract powder enhances the immunity of the COPD mice.

Table 9 immune organ index (mg/g) of mice in each group after administration

6.6 summary

(1) Regarding positive drugs

The main measures for treating chronic obstructive pulmonary disease at present are as follows: bronchodilators, including β2 receptor agonists, anticholinergic agents, and theophylline agents; glucocorticoids; phlegm-resolving drugs, etc.; wherein, the bronchodilator can well relieve COPD symptoms, but has no anti-inflammatory effect, and can treat both symptoms and root causes; the present application therefore aims to develop a safe and effective COPD anti-inflammatory drug. In order to have clear therapeutic indexes and measure the drug effect, the application selects the glucocorticoid drug dexamethasone which is marketed as a positive control, and compares the advantages and the disadvantages of the two drugs.

(2) Modeling feasibility of COPD in the application

The modeling method can be initially determined from the food intake, the weight gain rate and the systemic blood inflammation of mice in the modeling period to generate certain damage to the mice, and the pathological processes of airway obstruction, emphysema, inflammation and hypoimmunity of the mice can be proved by combining LPS with papain modeling through the subsequent staining of lung tissues HE, lung function respiration index, blood and BALF inflammatory cell measurement, inflammatory factor IL-6, TNF-alpha measurement and immune organ index measurement, which are similar to clinical COPD symptoms, so that the COPD modeling method is feasible.

(3) The Tibetan medicine compound treatment effect in the application

From the results of inflammatory cells in blood, bronchoalveolar lavage fluid (BALF) and inflammatory factor measurement of lung tissue, the model mice developed obvious inflammatory phenomena from local lung tissue to whole body. Compared with a model mouse, the Tibetan medicine compound medicine has obvious improvement phenomenon on various inflammation indexes. Compared with model mice, the Tibetan medicine compound medicine group has certain treatment effects on respiratory obstruction, emphysema and hypoimmunity, and the medicine has dose-dependent treatment effects. In addition, from the above results, it is clear that DEX has a good anti-inflammatory effect, but causes a serious decrease in immune function, possibly associated with side effects of hormonal drugs.

Example 7

In order to further define the anti-inflammatory mechanism of the Tibetan medicine composition, the application constructs different in-vitro inflammation models and observes the anti-inflammatory protection and the regulation effect of each inflammatory channel protein of the medicine. Macrophages play an important role in respiratory inflammatory diseases, while the most common inducer of macrophage activation is lipopolysaccharide LPS, LPS-treated RAW264.7 macrophages have been widely used as a model for studying inflammatory responses in vitro. A549 belongs to alveolar type ii epithelial cells, and often adopts LPS or CSE to stimulate the a549 to generate inflammatory response as an in vitro model for pulmonary disease research. Therefore, the application adopts LPS to induce macrophage RAW264.7 and alveolar II type epithelial cell A549 to construct the lung disease inflammation model.

Autophagy, a cellular waste removal system, plays a key role in regulating inflammatory responses. The AMPK/mTOR pathway is an important autophagy regulating pathway, when cells are stimulated by the outside, AMPK is activated and generates phosphorylation reaction, so that downstream mTOR gene phosphorylation reaction can be inhibited, autophagy cascade reaction is promoted, and activation of autophagy can inhibit inflammatory reaction by reducing secretion of pro-inflammatory cytokines. In addition, many studies have shown that oxidative stress inducers such as cigarette smoke, LPS, etc. can enhance inflammatory effects by modulating the MAPK pathways such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p 38 MAPK). The present application therefore demonstrates the anti-inflammatory mechanism of the compound composition by observing the protein modulating effects of the AMPK/mTOR pathway and the MAPK pathway.

7.1RAW264.7 cell culture and treatment

RAW264.7 cells were purchased from ATCC cell bank (USA) and grown in culture in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, penicillin 100U/ml and streptomycin 100. Mu.g/ml, incubator conditions 37℃and 5% CO2. The MTT method is adopted to measure the influence of Tibetan medicine compositions with different concentrations on RAW264.7 cell viability; cells were seeded in 96-well plates (1X 104 cells per well), pretreated with the appropriate concentration of drug after cell attachment, stimulated with LPS (1. Mu.g/ml) after 24 hours, and supernatants were collected 24 hours to determine inflammatory factors such as IL-6, IL-1. Beta., TNF-alpha.

7.2A549 cell culture and treatment

A549 cells were derived from university of eastern and grown in high-sugar DMEM medium containing 10% (v/v) fetal bovine serum, penicillin 100U/ml and streptomycin 100 μg/ml, with incubator conditions of 37 ℃,5% co2. The MTT method is adopted to measure the influence of Tibetan medicine compositions with different concentrations and LPS on the activity of A549 cells, and the protection effect of the medicine on LPS stimulation A549 at different time points.

7.3Western Blot

Cells were seeded in 6-well plates (2X 105/well), pre-treated with Tibetan drug composition after 24 hours, and then after 24 hours of stimulation of cells with lipopolysaccharide (LPS; sigma) (1. Mu.g/ml), washed 3 times with pre-chilled PBS, added with pre-chilled RIPA lysate containing protease inhibitor and phosphatase inhibitor, lysed on ice for 20min, centrifuged at 12000r for 10 minutes to obtain supernatant, and BCA protein was quantified to 5. Mu.g/. Mu.l. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE), transferred to PVDF for 1.5h, membrane was blocked in 5% skim milk formulated in TBST for 2h, membrane washed three times for 5 min/time, incubated overnight with primary antibody (1:1000) at 4℃for three times for 10 min/time. Incubation with anti-HRPP conjugated rabbit or IgG secondary antibody (1:3000) for 1h at room temperature, washing the membrane three times for 10 min/time. The proteins were visualized using a CCD system (Tanon 5200, china) by ECL solution development.

7.4 results

(1) The Tibetan medicine composition plays an anti-inflammatory role by activating the AMPK/mTOR autophagy pathway

As shown in figure 3, the application uses 1 mug/ml LPS to stimulate RAW264.7 to construct an inflammation model, and selects 0.5 mg/ml Tibetan medicine composition to intervene, and measures the level of inflammatory factors and autophagy protein content index after 24 hours. The results show that the secretion of IL-1 beta, IL-6 and TNF-alpha in RAW264.7 cells is obviously improved after LPS treatment, and the Tibetan medicine composition can inhibit the expression and release of the inflammatory factors in a dose-dependent manner. The immunoblotting result shows that the medicine can obviously increase the phosphorylation of AMPK, reduce the phosphate of mTOR, and further enhance autophagy to exert anti-inflammatory effect.

(2) The Tibetan medicine composition plays an anti-inflammatory role by inhibiting MAPK pathway activation

According to the cell viability result shown in fig. 4, the application selects 1 mug/ml LPS to stimulate A549 to construct an inflammation model, and selects 50 mug/ml Tibetan medicine composition and 100 mug/ml Tibetan medicine composition to intervene, and the protein content index is measured after 24 hours. The result shows that ERK, JNK, P protein of the COPD model group is obviously phosphorylated, and the downstream inflammatory proteins iNOS and COX2 content are obviously increased; the medicine group has obvious inhibiting effect on the activation state of MAPK channel protein, and can reduce the expression quantity of inflammatory protein and exert anti-inflammatory effect.

7.5 discussion and summary

The application induces RAW264.7 and A549 cells to generate inflammatory response through LPS, and the application has good anti-inflammatory effect after administration, is consistent with the in vivo research result, and has the regulation effect on AMPK/MTOR pathway and MAPK pathway proteins. Inflammatory factors such as IL-1 beta, IL-6 and TNF-alpha appear in the occurrence and development process of chronic inflammation of COPD, and excessive inflammatory factors can activate NF- κB complex, thereby promoting the expression of more inflammatory genes and amplifying inflammatory reaction. Therefore, the application selects the release amount of inflammatory factors to measure the anti-inflammatory effect of the medicine on macrophages. In addition, inducible NO synthase iNOS is the primary enzyme catalyzing the production of NO under inflammatory conditions, while excessive NO may promote the production of cytokines and matrix metalloproteinases, mitochondrial dysfunction and apoptosis, thereby accelerating the development of inflammation; cyclooxygenase-2 (COX-2) promotes Prostaglandin (PGs) production and plays an important role in chronic inflammation. Therefore, the application selects the expression quantity of iNOS and COX2 inflammatory proteins and measures the anti-inflammatory protection effect of the medicine on alveolar cells. It was found that activation of AMPK can ameliorate chronic inflammation and injury of COPD or emphysema by alleviating inflammatory response, slowing cellular senescence, inducing autophagy, and regulating mitochondrial metabolism; the biological anti-inflammatory activity research results of a plurality of medicinal plants show that the biological characteristics of the medicinal plants are exerted by blocking two main signal paths of NF- κB and mitogen-activated protein kinase MAPK, and the MAPK paths are shown to play a main role in the generation of various pro-inflammatory mediators. Thus, the present application selects the AMPK/MTOR pathway and MAPK pathway, demonstrating the anti-inflammatory mechanism of action of the compound composition. The above results indicate that the composition may exert autophagy-regulating effects by inhibiting MTOR phosphorylation by activating AMPK, and may affect inflammatory protein expression and inflammatory factor release by inhibiting MAPK pathway phosphorylation.

Example 8

In order to ensure the safety of the Tibetan medicine compound clinical application, the embodiment uses the maximum dosage method to carry out single administration on SD male and female rats, the medicine extract powder prepared by the method of the embodiment 2 is administrated by stomach irrigation, the acute toxic reaction condition generated after administration is observed, and the information of toxic reaction time and symptoms, target organs, death time, recovery time, animal weight change condition, death condition and the like is recorded, thereby providing a basis for determining the safety range and the toxic characteristics of the tested medicine.

8.1 grouping of animals

The quarantine period of the embodiment is 14 days, weighing is carried out on the last day of the quarantine period, and the weight of the rats is divided into 4 groups randomly according to layers of weights, and each of the SD male and female rats is divided into a control group and an administration group; 12 control groups, namely male and female halves; group 21, 10 females and 11 males were administered.

8.2 toxicity experiment development

According to the dissolution condition when preparing the liquid medicine and the difficulty of sucking the liquid medicine by a 16-gauge stomach needle, the maximum administration concentration of the extract powder of the Tibetan medicine composition is 1.5g/ml, and the single maximum administration volume is 20ml/kg, so that the single maximum administration amount is 30g/kg, which is equivalent to 210g crude drug/kg; the administration route is defined as gastric lavage administration;

the test uses a single maximum dose method, the administration group is administered once a day, the total dose is 30g/kg (292 times of the clinically effective dose), and the negative control group is administered with equal volume of distilled water. Two weeks after administration was observed continuously, and the results were determined by dissecting the material. One male rat of the administration group is anesthetized and killed in the dissection process, blood and viscera are not taken, but the viscera are observed visually to be free from lesions; one dosing group female rat had failed to take blood, had taken the viscera, and was observed visually for no lesions.

8.3 Experimental criteria

(1) Weight detection

Animal body weight was measured on the day of dosing, and on days 1, 2, 3, 5, 7, 10 and 14 after dosing, respectively.

(2) Index of blood chemistry

After 14 days after administration, the rat is subjected to abdominal aorta blood collection, and various biochemical indexes in the blood are detected, wherein the indexes include AST/ALT (glutamic oxaloacetic transaminase/glutamic pyruvic transaminase), BUN (blood urea nitrogen), TBI (total bilirubin) and TP (total protein), and the health conditions of the liver, the kidney, the gall bladder and the like and the whole body nutrition and health state can be detected.

(3) Blood physiological index

The rats were subjected to abdominal aortic blood collection 14 days after administration, and the white blood cell count, the red blood cell count, the hemoglobin concentration, and the platelet count in the blood were statistically analyzed using an automatic blood analyzer. Can be used for detecting inflammation, hematopoiesis, oxygen carrying capacity and blood coagulation. The health condition of the rats is comprehensively measured.

(4) Index of vital organ

The important organs such as heart, liver, spleen, lung, kidney, brain and the like were taken 14 days after administration, and the organ index was measured by weighing. In the process of dissection, whether obvious organ lesions exist or not is observed, so that whether organ damage risks exist in a large amount of the Tibetan medicine compound.

8.4 experimental results

(1) Weight gain rate

Female rats showed significant weight loss on the first day after dosing, recovery of weight gain was initiated on the second day, the rate of gain was slower than that of control group on the third day, and there was no significant difference between the data of starting weight on the fourth day and the control group; the weight of the male rats was extremely reduced on the first day after administration, and the male rats gradually recovered to normal on the second day without significant difference from the control group. The specific data are shown in tables 10 and 11. From the above, the weight increase rate index of the Tibetan medicine composition extract powder administered to the female and male rats at the maximum dose is not abnormal.

TABLE 10 female rat weight gain rate

Note that: compared with the control group, P <0.01 and P <0.05

TABLE 11 weight gain rate in Male rats

Note that: compared with the control group, P <0.01 and P <0.05

(2) Index of blood chemistry

Compared with the control group, the total protein TP index of the male rat administration group is obviously lower, but the average value is not different, the index belongs to normal values, and other indexes have no obvious difference. The blood biochemical index of female rats has no significant difference, and the specific results are shown in Table 12 and Table 13. From the above, it is apparent that the blood biochemical index is not abnormal after the Tibetan medicine composition extract powder is administered to the female and male rats at a maximum dose.

TABLE 12 Biochemical index of female rats

TABLE 13 Biochemical index of male rats

Note that: compared with the control group, P <0.01 and P <0.05

(3) Blood physiological index

There were no significant differences in blood normals including white blood cell WBC, red blood cell RBC, hemoglobin HGB, platelet PLT for both the female and male rat dosing groups compared to the control group. The specific results are shown in Table 14, table 15. From the above, it can be seen that: after the Tibetan medicine composition extract powder is administrated to female and male rats at a maximum dose, the blood physiological index is not abnormal.

TABLE 14 blood routine index for female rats

TABLE 15 blood routine index for Male rats

(4) Index of vital organ

There was no significant difference in visceral index in both the female and male rat dosing groups compared to the control group. The specific results are shown in Table 16, table 17. From the above, it can be seen that: after the Tibetan medicine composition extract powder is administrated to female and male rats at a maximum dose, the organ index is not abnormal.

TABLE 16 index of female rat organ (mg/g)

Table 17 index of Male rat organ (mg/g)

8.5 summary

The application evaluates the systemic and organ toxicity effects of the Tibetan medicine compound according to indexes such as the weight growth rate, the index of vital organs, hematology, hematochemistry and the like of rats. The result shows that the single large-dose gastric lavage Tibetan medicine composition extract suspension has a short-term and slight slowing effect on the weight growth of male and female rats, and gradually returns to normal after one day; the indexes of organs such as heart, liver, lung, kidney, brain and the like, such as hematology, hematochemistry and the like show that the indexes have no obvious abnormality, and no definite tissue damage is found during pathological anatomy. Thus, it was demonstrated that the Tibetan medicine composition of the present application had a single maximum dose of 30g/kg, and no obvious toxic symptoms were observed during the whole test period, which was 292 times the clinically intended dose.

Claims

1. The Tibetan medicine composition for treating the chronic obstructive pneumonia is characterized by being prepared from the following raw materials in parts by weight: 120-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of common knotweed herb, 19-25 parts of nutmeg, 50-60 parts of limestone, 20-30 parts of slag tamarind paste and 75-85 parts of liquorice.

2. The Tibetan medicine traditional Chinese medicine composition for treating chronic obstructive pneumonia according to claim 1, wherein the composition is prepared from the following raw materials in parts by weight: 140-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of common knotweed herb, 19-23 parts of nutmeg, 50-55 parts of limestone, 26-30 parts of slag tamarind paste and 75-79 parts of liquorice.

3. The Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 1 or 2, wherein the Tibetan medicine composition is prepared into a Tibetan medicine compound preparation by adding pharmaceutically acceptable auxiliary materials.

4. The Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 3, wherein the preparation is one of a granule, a tablet and a capsule.

5. A method for preparing a Tibetan medicine composition for treating chronic obstructive pulmonary disease according to claim 1 or 2, comprising the steps of: taking gentian flower, ash bark, common lantain herb, nutmeg and liquorice according to a certain proportion, soaking the gentian flower, ash bark, common lantain herb, nutmeg and liquorice in water, decocting or heating and refluxing for extraction, filtering, concentrating filtrate to obtain concentrated extract, adding slag tamarind paste, mixing, precipitating with ethanol, concentrating supernatant, mixing with limestone, drying, crushing and sieving to obtain extract powder.

6. The method for preparing a Tibetan medicine composition for treating chronic obstructive pulmonary disease according to claim 5, wherein the soaking time is 30-60min.

7. The method of claim 5, wherein the water is 8-14 times the weight of the medicinal materials for each decoction or extraction for 1-2 hours.

8. The method of claim 5, wherein the ethanol precipitation is performed by adding 3-4 times of 95% ethanol, and the ethanol precipitation time is 24 hours.

9. Use of a Tibetan medicine composition according to claim 1 or 2 in the preparation of a medicament for treating chronic obstructive pulmonary disease.