A Tibetan medicine composition for treating chronic obstructive pneumonia, its preparation method and application

Abstract

The present invention discloses a Tibetan medicine composition for treating chronic obstructive pneumonia, its preparation method and application. The Tibetan medicine composition is obtained by repeatedly optimizing the drug composition and content through clinical and basic research, and is made of seven medicinal materials including Gentiana scabra Bunge flower, Cortex Fraxini, Lagotis glauca, Myristica fragrans Houtt., Glycyrrhiza uralensis Fisch., Concretio Silicea Bambusae, and Zhaxun ointment. The Tibetan medicine composition of the present invention can reduce the expression levels of inflammatory factors in the serum and lung tissues of mice with chronic obstructive pneumonia efficacy models and improve lung function indicators, induce autophagy by activating the AMPK / mTOR pathway to reduce airway inflammation, and reduce the expression of inflammatory proteins by inhibiting the activation of MAPK pathway proteins. This formula is clinically applicable to the treatment of chronic obstructive pulmonary disease, is safe, effective, and quality controllable, and has better curative effects than chemical drugs such as glucocorticoids already on the market, and is a national medicine variety urgently needed in clinical practice.

Description

Technical Field

[0001] The invention belongs to the technical field of Tibetan medicine, and particularly relates to a Tibetan medicine composition for treating chronic obstructive pulmonary disease, a preparation method thereof and an application thereof. Background Art

[0002] Chronic Obstructive Pulmonary Disease (COPD for short) is a disease state characterized by irreversible airflow limitation. The airflow limitation develops repeatedly and progressively, and is related to the abnormal inflammatory response of the lungs to harmful gases or harmful particles; the exact cause of COPD is still unclear, which is similar to emphysema. Clinically, when patients with chronic bronchitis or (and) emphysema show airflow limitation during pulmonary function examination and the limitation is not completely reversible, they are diagnosed with COPD. Chronic bronchitis refers to the chronic and non-specific inflammation of the bronchial wall; emphysema refers to the abnormal and persistent dilation of the air cavities at the distal end of the terminal bronchioles in the lungs, accompanied by the destruction of the alveolar wall and bronchioles. In addition, COPD is a consumption to the body, which can damage the immunity and make patients prone to infectious diseases. Severe cases can lead to complications of other organs, such as cor pulmonale and pulmonary encephalopathy, resulting in serious damage to the heart and brain, and seriously threatening people's physical health and quality of life. With the development of society, the air pollution is getting worse, the haze environment is increasing, smoking and other pulmonary diseases are increasing, so that the incidence of COPD shows an upward trend year by year. At present, there is no effective treatment drug clinically, and patients cannot get timely prevention and treatment, resulting in a low disease control rate and a high mortality rate of patients. It is one of the diseases that endanger the health of the people in society.

[0003] The pathogenesis of COPD is complex and is related to chronic inflammation, oxidative stress, protease / antiprotease imbalance and immune imbalance. The clinical manifestations are chronic cough, expectoration, shortness of breath, dyspnea and other symptoms. The conventional treatment is mainly to relieve cough, expectorate phlegm and anti-inflammation. The main measure is to use bronchodilators. When the condition is severe, glucocorticoids are often combined with low-concentration oxygen inhalation; bronchodilators can better relieve the symptoms of COPD, but have no obvious anti-inflammatory effect and only treat the symptoms rather than the root cause; although glucocorticoids have good anti-inflammatory effects, long-term use has large side effects, and the body will produce glucocorticoid resistance, making the condition more serious. Traditional Chinese medicine has rich experience in the treatment of lung diseases. It has important scientific significance to search for drugs for treating COPD from the classic prescriptions of traditional Chinese medicine and ethnic medicines. Summary of the Invention

[0004] The purpose of the present invention is to find a suitable drug for treating COPD from classic Tibetan medicine prescriptions, carry out modification and optimization on this basis, explore new clinical indications of traditional Tibetan medicine for treating COPD, and develop a suitable pharmaceutical dosage form, so as to achieve the purpose of clear curative effect, increased patient compliance and safe medication.

[0005] To achieve the above object, the preparation scheme of the Tibetan medicine composition provided by the present invention is as follows:

[0006] A Tibetan medicine composition for treating chronic obstructive pneumonia, which is made from the following raw materials by weight: 120-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of lagotis, 19-25 parts of nutmeg, 50-60 parts of travertine, 20-30 parts of zha xun paste, 75-85 parts of liquorice.

[0007] Preferably, the above composition is made from the following raw materials by weight: 140-160 parts of gentian flower, 50-55 parts of ash bark, 50-55 parts of lagotis, 19-23 parts of nutmeg, 50-55 parts of travertine, 26-30 parts of zha xun paste, 75-79 parts of liquorice.

[0008] The above composition is prepared into a Tibetan medicine compound preparation by adding pharmaceutically acceptable excipients. The preparation can be one of granules, tablets, and capsules.

[0009] The preparation method of the above Tibetan medicine composition for treating COPD includes the following steps: taking gentian flower, ash bark, lagotis, nutmeg, and liquorice herbs in proportion, soaking in water and then decocting or heating under reflux for extraction, filtering, concentrating the filtrate to obtain a concentrated extract, adding zha xun paste and mixing, then performing alcohol precipitation, taking the supernatant and concentrating it, then combining it with travertine, drying, pulverizing, and sieving to obtain an extract powder.

[0010] The above soaking time in water is 30-60 min, and preferably the soaking time is 30-40 min.

[0011] The above decocting or heating under reflux is performed 2-3 times, and the decocting or extraction time each time is 1-2 hours, and the water consumption for each decocting or extraction is 8-14 times the weight of the herbs, preferably 8-12 times the weight.

[0012] The above alcohol precipitation is to add 3-4 times the volume of 95% ethanol, and the alcohol precipitation time is 24 hours.

[0013] The Tibetan medicine compound provided by the present invention can significantly improve the symptoms of COPD, and the application of the above Tibetan medicine composition in the preparation of drugs for treating chronic obstructive pneumonia has a significant curative effect.

[0014] The specific efficacy of the drug components used in this invention is as follows:

[0015] Gentian flower: This product is the dried flowering aerial part of Gentian veitchiorum Hemsl. of the Gentianaceae family. Identification basis: It conforms to the provisions under Gentiana biflora on page 98 of the first volume of the "Tibetan Medicine Material Standard of Tibet Autonomous Region" in 2004. It is recorded in "Jingzhu Materia Medica" that Tibetan medicine uses gentian flower as "Bangjian Wenbao" and can treat toxic diseases, various heat syndromes, and throat heat and obstruction.

[0016] Cortex Fraxini: This product is the dried branch bark or stem bark of Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb, Fraxinus szaboana Lingelsh., or Fraxinus stylosa Lingelsh. of the Oleaceae family. It complies with the regulations under Cortex Fraxini on page 282 of Part I of the Chinese Pharmacopoeia (2020 Edition). It is recorded in Compendium of Chinese Materia Medica that the functions and indications of Cortex Fraxini are to clear heat and dry dampness, clear the liver and improve eyesight, relieve cough and asthma. It is mainly used for damp-heat diarrhea, leukorrhea, red and swollen eyes, corneal nebula, and cough and asthma due to lung heat.

[0017] Lagotis Root: This product is the dried whole herb of Lagotis brevituba Maxim. or Lagotis integra W.Smith. of the Scrophulariaceae family. It is recorded in Flora of China that Lagotis root can treat lung abscess with cough and dyspnea, pulmonary tuberculosis, fullness in the chest with expectoration of purulent blood, pyogenic cavity due to blood heat, and scrofula, etc., and has the effects of clearing heat and removing fire, and anti-inflammatory and detoxifying.

[0018] Nutmeg: This product is the dried kernel of Myristica fragrans Houtt. of the Myristicaceae family. It is recorded in the Chinese Pharmacopoeia that the functions and indications of nutmeg are to warm the middle-jiao and promote qi movement, astringe the intestine and stop diarrhea. It is used for spleen-stomach deficiency-cold, chronic diarrhea, abdominal distension and pain, anorexia and vomiting.

[0019] Licorice Root: This product is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L. of the Leguminosae family. It is recorded in the Chinese Pharmacopoeia that the functions and indications of licorice root are to invigorate the spleen and replenish qi, clear heat and detoxify, resolve phlegm and relieve cough, relieve spasm and pain, and harmonize various medicines. It is used for spleen-stomach weakness, lassitude and weakness, palpitation and shortness of breath, cough with profuse phlegm, abdominal and limb spasmodic pain, carbuncles and sores, and to relieve the toxicity and potency of drugs.

[0020] Travertine: This product is a carbonate mineral, mainly containing calcium carbonate (CaCO3). It is recorded in the Compendium of Chinese Herbal Medicines that travertine has the effect of clearing heat and tonifying the lung, and is suitable for treating various lung heat diseases.

[0021] Zhaxun Extract: This product is a concentrated extract made by decocting Zhaxun with water and concentrating. It is recorded in the Tibetan medical work Four Medical Tantras that Zhaxun extract can cure all heat syndromes, especially has significant effects on clearing stomach heat, liver heat, and kidney heat, etc.

[0022] The Four Continuations, the foundational classic of Tibetan medicine, points out that the bodies of all living beings are formed by the five elements (earth, water, fire, wind, and space). The lungs belong to the Bacon area, and the five elements are earth and water, which are cold and have the function of regulating water and secreting mucus. Due to the characteristics of Bacon's cold and dampness and its distribution on the skin and orifices of the body surface, the respiratory tract is susceptible to external factors, causing symptoms such as coughing, coughing up thick sputum, and fever and inflammation. If Bacon is disturbed, it will become Bacon evil, which will cause the properties of Tripa such as heat, sharpness, lightness, and greasy to change and cause Tripa disease, resulting in heat syndrome, plague, and various inflammations. Long-term non-healing or repeated attacks will develop into chronic bronchitis. When the five elements in the human body are clinically diagnosed to be out of balance, drugs of different properties and effects will be used for treatment. For this disease, cold and cool drugs are generally used for treatment. There are many famous prescriptions for the treatment of respiratory diseases that are clearly recorded in the "Four Medical Classics", such as Fifteen-Ingredient Longdan Flower Pills, Nine-Ingredient Lime Hua powder, Three-Ingredient Longdan Flower Pills, Six-Ingredient Clove Pills, etc. The above Tibetan medicine prescriptions are clinically suitable for the treatment of bronchitis, senile asthma and other diseases. The medicinal taste is complex and there is no clinical use for the treatment of chronic obstructive pneumonia.

[0023] The present invention uses gentian flower as the main drug to reasonably formulate a prescription, adds cold Tibetan medicine that can cure lung heat to enhance the efficacy of gentian flower, and is compatible with corresponding medicines that can prevent bacon syndrome or lung syndrome caused by cold medicines, so as to achieve the purpose of achieving a coordinated state of the three factors of lung, red bark and bacon. The specific formula is that gentian flower is the main drug, Qinpi helps gentian flower to clear heat and dry dampness; calcite helps gentian flower to clear heat and nourish the lung; rabbit ear grass and dregs of paste help gentian flower to clear heat and detoxify, among which rabbit ear grass can not only prevent bacon syndrome, but also clear lung and stomach congestion and discharge pus; warm and spicy medicine nutmeg prevents lung syndrome, warms the middle and promotes qi, and regulates the symptoms of spleen and stomach deficiency and cold; and liquorice is used to harmonize various medicines, nourish the spleen and replenish qi, and relieve pain. It has been proved by the optimization of Tibetan medicine clinical trials and modern pharmacology that this prescription is suitable for treating chronic obstructive pneumonia.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] 1. The Tibetan medicine composition of the present invention has reasonable compatibility and is an empirical prescription formulated by the inventor based on the theoretical knowledge of Tibetan medicine combined with many years of clinical practice.

[0026] 2. The Tibetan medicine composition of the present invention has fewer medicinal flavors and a simple process, which is beneficial to drug production and quality control.

[0027] 3. The Tibetan medicine composition of the present invention is mainly made of seven medicinal materials, namely gentian flower, fraxinus, lagotis glauca, nutmeg, liquorice, travertine, and zha xun paste, and is obtained by repeatedly optimizing the drug composition and ratio through clinical and basic research. The Tibetan medicine composition can reduce the expression levels of inflammatory factors in the serum and lung tissues of mice with a chronic obstructive pneumonia pharmacodynamic model and improve lung function indicators. It can induce autophagy by activating the AMPK / mTOR pathway to reduce respiratory tract inflammation, and reduce the expression of inflammatory proteins by inhibiting the activation of MAPK pathway proteins. Therefore, based on a large amount of animal experiment research data, the Tibetan medicine composition can improve lung respiratory function, exert anti-inflammatory effects, enhance immunity, and has no obvious toxicity and adverse reactions. This prescription is clinically applicable to the treatment of respiratory inflammatory diseases such as chronic obstructive pulmonary disease, is safe, effective, quality controllable, and has better curative effects than chemical drugs such as glucocorticoids already on the market, and is a national medicine variety urgently needed in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 It is a pathological section diagram (×100) of HE staining of the lung tissue of COPD mice by the Tibetan medicine composition described in the present invention;

[0030] Figure 2 It is a pathological section diagram (×200) of HE staining of the lung tissue of COPD mice by the Tibetan medicine composition described in the present invention;

[0031] Figure 3 It is a mechanism diagram of the respiratory tract inflammation protection effect of the Tibetan medicine composition described in the present invention on macrophages Raw264.7 by activating the AMPK / mTOR autophagy pathway; in the figure, (A) is a bar graph of the effects of different concentrations of LDZK on the viability of Raw264.7 cells; (B) is the release amount of the inflammatory factor IL-1β in each group of cells; (C) is the release amount of the inflammatory factor IL-6 in each group of cells; (D) is the release amount of the inflammatory factor TNF-α in each group of cells; (E) is the original strip diagram of the AMPK / mTOR pathway protein;

[0032] Figure 4 It is a mechanism diagram of the respiratory tract inflammation protection effect of the Tibetan medicine composition described in the present invention on alveolar epithelial cells A549 by inhibiting the activation of the MAPK pathway; in the figure, (A) is the toxicity study of different concentrations of LPS on A549 cells; (B) is a bar graph of the effects of different concentrations of LDZK on the viability of A549 cells; (C) is the protective effect of LDZK on the cytotoxicity induced by LPS at different time points (12, 24, 48h); (D) is the original strip diagram of the inflammatory proteins INOS, COX2, and MAPK pathway proteins. Specific implementation method

[0033] The present invention will be further clarified below in conjunction with specific embodiments. The following embodiments are only used for detailed explanation of the present invention and not for limiting the scope of the present invention; in addition, it should be understood that after reading the content of the present invention, those skilled in the art's various equivalent modifications of the present invention all fall within the scope defined by the appended claims of this application.

[0034] Example 1

[0035] Take 55 g of travertine, crush it, and sieve it (80 mesh), and set aside. Weigh 120 g of the raw medicine Gentiana scabra, 55 g of Cortex Fraxini, 55 g of Lagotis glauca, 23 g of Myristica fragrans, and 80 g of Glycyrrhiza uralensis. Remove impurities, cut them into pieces and place them in an extraction device. Add 10 times the amount of water of the raw medicine. After soaking for 30 min, heat and decoct for 1 h, filter with a 100-mesh filter cloth to obtain the first extraction solution. Then add 8 times the amount of water of the raw medicine, heat and decoct for 1 h, filter with a 100-mesh filter cloth to obtain the second extraction solution. Combine the two extraction solutions, concentrate under reduced pressure to obtain a concentrated extract, add 26 g of Zhaxun extract, mix evenly, and add 3 times the volume of 95% ethanol of the extract mixture. Stir evenly and then stand for alcohol precipitation for 24 h. Take the supernatant and concentrate it, combine it with travertine and dry it, crush and sieve to obtain an extract powder. Then add conventional doses of microcrystalline cellulose and lactose, mix and granulate by a conventional method with 75% ethanol, and sieve to prepare granules.

[0036] Example 2

[0037] Take 50 g of travertine, crush it, and sieve it (80 mesh), and set aside. Weigh 160 g of the raw medicine Gentiana scabra, 50 g of Cortex Fraxini, 50 g of Lagotis glauca, 19 g of Myristica fragrans, and 75 g of Glycyrrhiza uralensis. Remove impurities, cut them into pieces and place them in an extraction device. Add 12 times the weight of water of the raw medicine. After soaking for 30 min, heat and decoct for 2 h, filter with a 100-mesh filter cloth to obtain the first extraction solution. Then add 10 times the amount of water of the raw medicine, heat and decoct for 1 h, filter with a 100-mesh filter cloth to obtain the second extraction solution. Combine the two extraction solutions, concentrate under reduced pressure to obtain a concentrated extract, add 30 g of Zhaxun extract, mix evenly, add 3 times the volume of 95% ethanol of the extract mixture. Stir evenly and then stand for alcohol precipitation for 24 h. Take the supernatant and concentrate it, combine it with travertine and dry it, crush and sieve to obtain an extract powder. Then add conventional doses of starch, powdered sugar, and dextrin, and granulate by a conventional wet granulation method. Mix it evenly with magnesium stearate and press tablets to prepare tablets.

[0038] Example 3

[0039] Take 52.5 g of travertine, crush it, sieve it (80 mesh), and set aside. Weigh 140 g of the crude drug Gentiana scabra Bunge, 50 g of Cortex Fraxini, 52.5 g of Lagotis glauca, 21 g of Myristica fragrans, and 79 g of Glycyrrhiza uralensis Fisch. Remove the impurities, cut them into pieces and place them in the extraction equipment. Add 10 times the weight of water of the crude drug, soak for 30 min, then heat and decoct for 1 h. Filter through a 100-mesh filter cloth to obtain the first extraction solution. Then add 8 times the weight of water of the crude drug, heat and decoct for 1 h, and filter through a 100-mesh filter cloth to obtain the second extraction solution. Combine the two extraction solutions, concentrate under reduced pressure to obtain a concentrated extract, add 28 g of Zhaxun extract, mix evenly, add 3 times the volume of 95% ethanol to the extract mixture, stir evenly and then let it stand for alcohol precipitation for 24 h. Take the supernatant and concentrate it, combine it with travertine and dry it, crush and sieve to obtain an extract powder, add a conventional dose of starch, fill into empty capsules, and prepare capsules.

[0040] Example 4

[0041] Take 52.5 g of travertine, crush it, sieve it (80 mesh), and set aside. Weigh 140 g of the crude drug Gentiana scabra Bunge, 52.5 g of Cortex Fraxini, 52.5 g of Lagotis glauca, 21 g of Myristica fragrans, and 77 g of Glycyrrhiza uralensis Fisch. Remove the impurities, cut them into pieces and place them in the extraction equipment. Add 10 times the amount of water of the crude drug, soak for 30 min, then heat and decoct for 1 h. Filter through a 100-mesh filter cloth to obtain the first extraction solution. Then add 10 times the amount of water of the crude drug, heat and decoct for 2 h, and filter through a 100-mesh filter cloth to obtain the second extraction solution. Combine the two extraction solutions, concentrate under reduced pressure to obtain a concentrated extract, add 28 g of Zhaxun extract, mix evenly, add 4 times the volume of 95% ethanol to the extract mixture, stir evenly and then let it stand for alcohol precipitation for 24 h. Take the supernatant and concentrate it, combine it with travertine and dry it, crush and sieve to obtain an extract powder, add a conventional dose of concentrated sucrose aqueous solution, and prepare a syrup.

[0042] Example 5

[0043] Take 55 g of travertine, crush it, sieve it (80 mesh), and set aside. Weigh 140 g of the crude drug Gentiana scabra Bunge, 52.5 g of Cortex Fraxini, 52.5 g of Lagotis glauca, 21 g of Myristica fragrans, and 77 g of Glycyrrhiza uralensis Fisch. Remove the impurities, cut them into pieces and place them in the extraction equipment. Add 12 times the amount of water of the crude drug, soak for 30 min, then heat and decoct for 1 h. Filter through a 100-mesh filter cloth to obtain the first extraction solution. Then add 10 times the amount of water of the crude drug, heat and decoct for 1 h, and filter through a 100-mesh filter cloth to obtain the second extraction solution. Combine the two extraction solutions, concentrate under reduced pressure to obtain a concentrated extract, add 28 g of Zhaxun extract, mix evenly, add 3 times the volume of 95% ethanol to the extract mixture, stir evenly and then let it stand for alcohol precipitation for 24 h. Take the supernatant and concentrate it, combine it with travertine, dry it, crush, sieve to obtain an extract powder, add a pharmaceutically acceptable wetting agent to obtain a mass, use a pill-making machine to roll it into pills, and prepare pills.

[0044] The above-mentioned Zhaxun Extract is a concentrated extract prepared by decocting Zhaxun with 5-10 times its weight of water for 1-2 times and then concentrating the filtrate.

[0045] Example 6

[0046] To study the efficacy of this Tibetan medicine compound on chronic obstructive pulmonary disease (COPD), the present invention established a mouse model of chronic obstructive pulmonary disease by instilling lipopolysaccharide (LPS) into the respiratory tract and atomizing papain, and carried out a pharmacodynamic experiment with this as the research object. The feasibility of the modeling method was initially judged by observing the body weight of the mice and the blood inflammation indexes during the modeling period. After the experiment, the feasibility of the COPD modeling method was finally judged by the pathological sections of the lung tissues of the model group and the control group, the lung respiratory function, the bronchoalveolar lavage fluid (BALF), the blood inflammation cell indexes, and the immune organ indexes, etc. And the therapeutic effect of this Tibetan medicine prescription on COPD was determined by comparing various indexes of each drug group and the model mice.

[0047] The following is a verification and description of the present invention through pharmacodynamic test examples:

[0048] Test drug (drug extract powder prepared by the method of Example 2), control drug (dexamethasone), lipopolysaccharide LPS, papain Papain, 60 male C57 mice, isoflurane, 25% urethane, nebulizer, blood analyzer, (EMMS) FM forced lung function detection system, tissue homogenizer, lipid oxidation (MDA) detection kit, Mouse IL-6 ELISA Kit, Mouse TNF-α ELISA Kit.

[0049] 6.2 Animal grouping

[0050] A total of 60 C57 mice were quarantined for 7 days and weighed on the last day of the quarantine period. 9 mice were randomly selected as the blank group according to body weight stratification; the remaining mice were modeled together, and the dead mice were excluded after modeling, and then randomly divided into COPD model group, dexamethasone (DEX) group, low-concentration Tibetan medicine group, and high-concentration Tibetan medicine group. There were 8-9 mice in each group.

[0051] 6.3 Model establishment and drug administration

[0052] Except for the normal control group, each of the other groups instilled 50 μl of LPS (1 mg / ml) into the nasal cavity on the 1st day of the experiment, and instilled 25 μl of LPS (1 mg / ml) into the nasal cavity on the 15th and 27th days. After 1-3 days of instilling LPS, papain (1 mg / ml) was atomized for 1 min per mouse, atomized every other day, and the model was established for 4 weeks.

[0053] After the model was established, drugs were administered. In the model group, 10 ml / kg of normal saline was administered by gavage; in the DEX group, 0.2 mg / kg of DEX solution was administered by gavage; in the low-concentration Tibetan medicine group, 102.75 mg / kg of the suspension of the drug extract powder was administered by gavage, corresponding to 0.9 g of crude drug / kg; in the high-concentration Tibetan medicine group, 205.5 mg / kg of the suspension of the drug extract powder was administered by gavage, corresponding to 1.8 g of crude drug / kg. Gavage was performed for a total of 4 weeks. In the first 2 weeks before the drug administration period, papain (1 mg / ml) was atomized every other day for 1 min per mouse. The total experimental period was 8 weeks.

[0054] After the gavage ended in the 8th week, 5 mice were selected from each group for pulmonary function measurement. After the measurement, the chest was opened to take lung tissues. The left lung was fixed in 4% paraformaldehyde, and the right lung was weighed and stored at -80 °C for later measurement of the kit indicators. In addition, blood was taken from the eyeballs of 3-4 mice in each group, and then bronchoalveolar lavage was performed to collect BALF fluid for determination of white blood cell differential count.

[0055] 6.4 Experimental indicators

[0056] (1) Body weight detection

[0057] The body weight of the mice was measured every week, and the activity status, food intake, etc. of the mice were observed.

[0058] (2) Blood inflammatory cell indicators

[0059] Blood was taken from the orbital cavities of the mice at the 4th and 8th weeks, and a blood automatic analyzer was used to statistically analyze the white blood cell count, neutrophil count, and lymphocytes in the blood.

[0060] (3) Inflammatory cell indicators in bronchoalveolar lavage fluid BALF

[0061] The skin at the throat of the mouse was cut open, and the tissues around the trachea were separated with forceps to expose the trachea. The tip of the 7-gauge needle of the syringe was cut off and then ground flat. The animal was fixed in the supine position. First, two surgical sutures were passed between the trachea and the esophagus, and a small scissors was used to make a transverse T-shaped incision at the thyroid position. The self-made needle was inserted into the trachea, and ligatures were tightened at the trachea where the cannula entered and at the distal end of the cannula. The self-made needle was connected to a 1-ml syringe to lavage 0.5 ml of normal saline, left for 10 s and then slowly withdrawn. This was repeated twice. Milky white foamy liquid could be seen. When it was felt that the piston could not be withdrawn with resistance, the lavage was stopped and transferred to an EP tube. The above lavage process was repeated 3 times. The collected liquid was considered a qualified lavage fluid if the recovery rate reached over 80%. A blood automatic analyzer was used to count the total number of white blood cells, neutrophils, lymphocytes, and eosinophils.

[0062] (4) Determination of IL-6 and TNF-α in lung tissues

[0063] Take the right lower lobe of the lung without pulmonary perfusion, weigh about 30 mg, add PBS according to the ratio of 1:9 for lung tissue homogenization, centrifuge at 12,000 r and 4 °C for 10 minutes, and take the supernatant. Detect IL-6 and TNF-α strictly according to the steps of the kit.

[0064] (5) Lung function indexes

[0065] After the end of the eighth week, measure the lung function of the mice. Fix the limbs and head of the anesthetized mice, disinfect the neck, bluntly separate the subcutaneous tissue of the neck layer by layer until the trachea is exposed and separated. Make a transverse incision at the upper segment of the tracheal ring, insert a tracheal intubation and ligate and fix it with surgical thread. Then put the mice into the mouse chamber of the lung function instrument, connect the small animal lung function instrument for mechanical ventilation measurement. The measurement results of each mouse are repeated three times. The lung function indexes include forced expiratory volume in 50 ms (FEV50), forced vital capacity (FVC), functional residual capacity of the lung FRC, peak expiratory flow PEF, and maximum mid-expiratory flow MMEF.

[0066] (6) Lung tissue pathological sections

[0067] Take the fixed left lung and process it according to the steps of dehydration, wax infiltration, embedding, sectioning, dewaxing to water, hematoxylin staining, eosin staining, dehydration and sealing. Finally, examine under a microscope and collect and analyze images at 100-fold and 200-fold fields of view.

[0068] (7) Immune organ indexes

[0069] After bronchoalveolar lavage of the mice and after measuring the respiratory function of the mice, take the thymus and spleen for weighing and measure the organ indexes. The immune and defense abilities of COPD patients against respiratory pathogenic microorganisms will decline, and thymus and spleen indexes are usually used to evaluate the host immune ability.

[0070] 6.5 Experimental results

[0071] (1) Modeling results

[0072] (a) General state of mice during the modeling period

[0073] Compared with the control group, the food intake of the model mice decreased, and the body weight decreased severely. The specific results are shown in Table 1. In addition, the model mice showed symptoms such as wheezing, head nodding breathing, deep and rapid breathing, abdominal muscle twitching, huddling and arching the back, and the spontaneous activity decreased, reaching a mortality rate of about 25%.

[0074] Table 1 Weight growth rate of mice in each group during the modeling period

[0075]

[0076] Note: Compared with the control group, **P<0.01, *P<0.05

[0077] (b) Inflammatory indicators of mice during the modeling period

[0078] As shown in the results analysis of Table 2, compared with the control group, the white blood cells in the blood of the model mice increased extremely significantly, and the neutrophils and lymphocytes also increased significantly, indicating that severe inflammatory reactions occurred in the mice and corresponding immune responses were induced.

[0079] Table 2 Number of inflammatory cells in each group of mice during the modeling period (10 9 / L)

[0080]

[0081] Note: Compared with the control group, **P < 0.01, *P < 0.05

[0082] (2) Treatment results

[0083] (a) Effect of the Tibetan medicine compound of the present invention on the general state of COPD mice

[0084] Compared with the control group, the weight growth rate and spontaneous activity of the other groups were significantly reduced; compared with the COPD model group, the spontaneous activity of the Tibetan medicine compound drug group of the present invention was high, the state was good, and the weight growth rate increased steadily; the weight of the model group mice showed a trend of increasing first and then decreasing, indicating that the late development of COPD still had a certain impact on the weight; the weight of the dexamethasone group showed a downward trend and the activity was not high. The specific weight results are shown in Table 3.

[0085] Table 3 Weight growth rate of each group of mice after administration

[0086]

[0087] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0088] (b) Effect of the Tibetan medicine compound of the present invention on blood inflammatory cells of COPD mice

[0089] Compared with the model group, the white blood cells WBC and lymphocytes Lym in the blood of the Tibetan medicine compound drug group of the present invention were significantly reduced in a dose-dependent manner, and the neutrophil index showed a dose-dependent decreasing trend. Compared with the model group, the WBC and Lym of the DEX group were significantly reduced, but the Neu index showed a significant increasing trend. The specific results are shown in Table 4, indicating that the Tibetan medicine compound extract powder has good systemic anti-inflammatory effects and is more stable in efficacy than DEX.

[0090] Table 4 Number of blood inflammatory cells in each group of mice after administration (10 9 / L)

[0091]

[0092] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0093] (c) Effects of the Tibetan medicine compound of the present invention on inflammatory cell indexes in BALF of COPD mice

[0094] Compared with the model group, the inflammatory white blood cells WBC in the alveolar lavage fluid of the Tibetan medicine compound group of the present invention showed a decreasing trend, and the high-concentration drug group showed an obvious decreasing trend; compared with the model group, the WBC index in the DEX group decreased significantly. Compared with the model group, the eosinophil counts in the BALF of the Tibetan medicine compound group and the DEX group decreased extremely significantly; the neutrophil counts showed a decreasing trend with no significant difference. The specific results are shown in Table 5, preliminarily indicating that both the Tibetan medicine compound and dexamethasone DEX have anti-inflammatory effects on the lungs.

[0095] Table 5 Inflammatory cell counts in BALF of mice in each group after administration (10 9 / L)

[0096]

[0097] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0098] (d) Effects of the Tibetan medicine compound of the present invention on IL-6 and TNF-α indexes in lung tissues of COPD mice

[0099] Compared with the model group, the inflammatory factor IL-6 and TNF-α indexes in the Tibetan medicine compound group of the present invention both showed a decreasing trend, among which the high-concentration drug group showed a significant decrease, and the inflammatory factor indexes in the DEX group also decreased significantly. The specific results are shown in Table 6. From these indexes, it is further indicated that the Tibetan medicine compound of the present invention has obvious anti-inflammatory effects on COPD mice and shows dose dependence, and its anti-inflammatory effect is comparable to that of the positive drug.

[0100] Table 6 Contents of IL-6 and TNF-α in lung tissues of mice in each group after administration (pg / mgprot)

[0101]

[0102] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0103] (e) Effects of the Tibetan medicine compound of the present invention on lung function indexes of COPD mice

[0104] Compared with the model group, the 50 - millisecond forced expiratory volume ((FEV50), forced vital capacity (FVC), maximum mid - expiratory flow (MMEF), and peak expiratory flow (PEF) of the Tibetan medicine compound group all showed an increasing trend. Among them, the high - concentration drug group increased significantly. The functional residual capacity (FRC) showed a decreasing trend, and the high - concentration drug group decreased significantly. Compared with the model group, the FEV50, MMEF, PEF, and FRC indexes of the dexamethasone (DEX) group all showed significant improvement, but the FVC index was worse than that of the model group. The specific results are shown in Table 7. From the above results, it can be seen that the LDZK drug has a dose - dependent preventive and therapeutic effect on COPD respiratory obstruction.

[0105] Table 7 Pulmonary function indexes of mice in each group after administration

[0106]

[0107] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0108] (f) Effect of the Tibetan medicine compound of the present invention on the pathological sections of the lungs of COPD mice

[0109] In order to quantify the pathological changes of lung tissue and more clearly and directly compare the results, referring to the literature, a scoring table for the pathological degree of lung tissue can be obtained, as shown in Table 8. The specific results of the pathological sections of lung tissue are shown in Figure 1 (×100), Figure 2 (×200).

[0110] Compared with the normal group, the alveolar morphology of the model group was abnormal, the structure was damaged, some alveolar cavities were fused into large bullae, and inflammatory cell infiltration was visible in the alveoli; there was a large amount of inflammatory cell infiltration in the lung interstitium, and the lung interstitium was significantly thickened; the bronchial wall was thickened, the lumen was narrowed, the epithelial cells of the bronchial wall showed necrosis, and cell debris and mucus appeared in the lumen. Compared with the model group, the alveolar damage and inflammatory cell infiltration in the DXM group and the low - concentration drug group were slightly improved, but the bronchial damage was not significantly improved; in the high - concentration drug group, most of the alveolar morphology was normal, some alveolar fusions were visible, and the inflammatory cell infiltration and bronchial damage were significantly improved.

[0111] Table 8 Scoring table for the pathological degree of lung tissue of mice in each group after administration

[0112]

[0113] (g) Effect of the Tibetan medicine compound of the present invention on the immune organ indexes of COPD mice

[0114] Compared with the normal group, the spleen index and thymus index of the model group and the Tibetan medicine compound drug group were both decreased, indicating that the establishment of the COPD model caused immune regulation imbalance. Compared with the model group, the spleen index and thymus index of the LDZK drug group increased in a dose-dependent manner; the spleen index and thymus index of the dexamethasone group were smaller than those of the model group. The specific results are shown in Table 9. This shows that dexamethasone severely decreased the body's immunity, while the LDZK lozenge extract powder enhanced the immunity of COPD mice.

[0115] Table 9 Immune organ indices of mice in each group after administration (mg / g)

[0116]

[0117] Note: Compared with the control group, **P < 0.01, *P < 0.05; compared with the COPD group, ##P < 0.01, #P < 0.05

[0118] 6.6 Summary

[0119] (1) Regarding the positive drug

[0120] The current main treatment measures for chronic obstructive pulmonary disease include: bronchodilators, including β2 receptor agonists, anticholinergic drugs, and theophylline drugs; glucocorticoids; expectorant drugs, etc. Among them, bronchodilators can well relieve the symptoms of COPD, but have no anti-inflammatory effect, treating the symptoms rather than the cause. Therefore, the present invention aims to develop a safe and effective anti-inflammatory drug for COPD. In order to have a clear treatment index to measure the drug effect, the present invention selects the marketed glucocorticoid drug dexamethasone as a positive control to compare the advantages and disadvantages of the two drugs.

[0121] (2) Regarding the feasibility of the COPD model establishment in the present invention

[0122] From the food intake, body weight growth rate, and systemic blood inflammation of the mice during the model establishment period, it can be preliminarily determined that this model establishment method caused certain damage to the mice. Through the later HE staining of lung tissue, pulmonary function respiratory indexes, determination of inflammatory cells in blood and BALF, determination of inflammatory factors IL-6 and TNF-α, and determination of immune organ indices, it can be clarified that the LPS combined with papain model establishment caused a pathological process of airway obstruction, emphysema, inflammation, and immune deficiency in mice, which is similar to the clinical COPD symptoms. This shows that this COPD model establishment method is feasible.

[0123] (3) Regarding the therapeutic effect of the Tibetan medicine compound in the present invention

[0124] From the results of measuring inflammatory cells in blood, bronchoalveolar lavage fluid (BALF), and inflammatory factors in lung tissue, it can be seen that obvious inflammatory phenomena occurred in the model mice from local lung tissue to the whole body. Compared with the model mice, various inflammatory indexes in the Tibetan medicine compound drug group showed obvious improvement. From the results of HE staining of lung tissue, respiratory indexes, and immune organ indexes, it can be known that compared with the model mice, the Tibetan medicine compound drug group had a certain therapeutic effect on respiratory obstruction, emphysema, and immune deficiency, and this drug showed a dose-dependent therapeutic effect. In addition, from the above results, it can be seen that the DEX drug had a good anti-inflammatory effect, but it would cause a serious decline in immune function, which might be related to the side effects of hormonal drugs.

[0125] Example 7

[0126] In order to further clarify the anti-inflammatory mechanism of this Tibetan medicine composition, the present invention constructed different in vitro inflammatory models to observe the anti-inflammatory protection of the drug and the regulatory effects of various inflammatory pathway proteins. Macrophages play an important role in respiratory inflammatory diseases, and the most common inducer of macrophage activation is lipopolysaccharide (LPS). RAW 264.7 macrophages treated with LPS have been widely used as a model for studying in vitro inflammatory responses. A549 belongs to type II alveolar epithelial cells, and LPS or cigarette smoke extract (CSE) is often used to stimulate A549 to produce an inflammatory response as an in vitro model for studying lung diseases. Therefore, the present invention used LPS to induce macrophages RAW264.7 and type II alveolar epithelial cells A549 to construct an inflammatory model of lung diseases.

[0127] Autophagy, as a cellular waste clearance system, plays a key role in regulating inflammatory responses. The AMPK / mTOR pathway is an important autophagy regulatory pathway. When cells are stimulated by the outside world, AMPK is activated and undergoes a phosphorylation reaction, which can inhibit the phosphorylation reaction of the downstream mTOR gene, and then promote the occurrence of the autophagy cascade reaction. Activating autophagy can inhibit inflammatory responses by reducing the secretion of pro-inflammatory cytokines. In addition, a number of studies have shown that oxidative stress inducers such as cigarette smoke and LPS can enhance inflammatory effects by regulating mitogen-activated protein kinase (MAPK) pathways such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK). Therefore, the present invention explained the anti-inflammatory mechanism of this compound composition by observing the protein regulatory effects of the AMPK / mTOR pathway and the MAPK pathway.

[0128] 7.1 RAW264.7 Cell Culture and Treatment

[0129] RAW 264.7 cells were purchased from the ATCC cell bank (USA) and cultured in RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin at 37°C and 5% CO2 in an incubator. The MTT method was used to determine the effects of the Tibetan medicine composition at different concentrations on the viability of RAW 264.7 cells; the cells were seeded in a 96-well plate (1×104 cells per well), and after cell adhesion, they were pretreated with the appropriate concentration of the drug. After 24 hours, the cells were stimulated with LPS (1 μg / ml), and after 24 hours, the supernatant was collected to measure inflammatory factors such as IL-6, IL-1β, and TNF-α.

[0130] 7.2 Culture and treatment of A549 cells

[0131] A549 cells were obtained from Southeast University and cultured in high-glucose DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin at 37°C and 5% CO2 in an incubator. The MTT method was used to determine the effects of the Tibetan medicine composition and LPS at different concentrations on the viability of A549 cells, and to determine the protective effect of the drug on LPS-stimulated A549 at different time points.

[0132] 7.3 Western Blot

[0133] The cells were seeded in a 6-well plate (2×105 / well). After 24 hours, they were pretreated with the Tibetan medicine composition, and then stimulated with lipopolysaccharide (LPS; Sigma) (1 μg / ml) for 24 hours. Then, they were washed 3 times with pre-cooled PBS, and pre-cooled RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added for lysis on ice for 20 min. The supernatant was collected by centrifugation at 12000 r and 4°C for 10 minutes, and the protein was quantified by BCA to 5 μg / μl. The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS / PAGE), transferred to PVDF for 1.5 h, and the membrane was blocked in 5% skim milk prepared with TBST for 2 h, washed three times for 5 min each time, incubated with the primary antibody (1:1000) at 4°C overnight, washed three times for 10 min each time. Incubated with anti-HRPp-conjugated rabbit or IgG secondary antibody (1:3000) at room temperature for 1 h, washed three times for 10 min each time. Developed with ECL solution, and the proteins were visualized using a CCD system (Tanon 5200, China).

[0134] 7.4 Results

[0135] (1) The Tibetan medicine composition of the present invention exerts an anti-inflammatory effect by activating the AMPK / mTOR autophagy pathway

[0136] As attached Figure 3, in this invention, an inflammation model was constructed by stimulating RAW264.7 with 1 μg / ml LPS, and a Tibetan medicine composition at 0.5 and 1 mg / ml was selected for intervention. After 24 h, the levels of inflammatory factors and the content of autophagy proteins were measured. The results showed that the secretion of IL-1β, IL-6, and TNF-α in RAW 264.7 cells was significantly increased after LPS treatment, and the Tibetan medicine composition could inhibit the expression and release of these inflammatory factors in a dose-dependent manner. The immunoblotting results showed that this medicine could significantly increase the phosphorylation of AMPK and reduce the phosphorylation of mTOR, thereby enhancing autophagy to exert an anti-inflammatory effect.

[0137] (2) The Tibetan medicine composition of this invention exerts an anti-inflammatory effect by inhibiting the activation of the MAPK pathway

[0138] Attached Figure 4 Based on the cell viability results, in this invention, an inflammation model was constructed by stimulating A549 with 1 μg / ml LPS, and a Tibetan medicine composition at 50 and 100 μg / ml was selected for intervention. After 24 h, the protein content index was measured. The results showed that the phosphorylation of ERK, JNK, and P38 proteins in the COPD model group was significantly increased, and the content of downstream inflammatory proteins iNOS and COX2 was significantly increased; the drug group had an obvious inhibitory effect on the activation state of these MAPK pathway proteins and reduced the expression of inflammatory proteins to exert an anti-inflammatory effect.

[0139] 7.5 Discussion and summary

[0140] In this invention, LPS is used to induce inflammatory responses in RAW264.7 and A549 cells. After drug administration intervention, good anti-inflammatory effects are shown, which is consistent with the in vivo research results, and it has a regulatory effect on the proteins of AMPK / MTOR pathway and MAPK pathway. Inflammatory factors such as IL-1β, IL-6, and TNF-α appear in the occurrence and development of chronic inflammation in COPD. Excessive inflammatory factors will activate the NF-κB complex, and then promote the expression of more inflammatory genes and amplify the inflammatory response. Therefore, in this invention, the release amount of inflammatory factors is selected to measure the anti-inflammatory effect of drugs on macrophages. In addition, inducible nitric oxide synthase iNOS is the main enzyme that catalyzes the production of NO under inflammatory conditions, and excessive NO may promote the production of cytokines and matrix metalloproteinases, mitochondrial dysfunction and apoptosis, thus accelerating the development of inflammation; cyclooxygenase-2 (COX-2) promotes the production of prostaglandins (PGs) and plays an important role in chronic inflammation. Therefore, in this invention, the expression levels of iNOS and COX2 inflammatory proteins are selected to measure the anti-inflammatory and protective effects of drugs on alveolar cells. It is found that the activation of AMPK can improve the chronic inflammation and damage of COPD or emphysema by reducing inflammatory responses, slowing down cell senescence, inducing autophagy and regulating mitochondrial metabolism, etc.; the research results of the biological anti-inflammatory activities of many medicinal plants show that they exert their biological properties by blocking two main signaling pathways, NF-κB and mitogen-activated protein kinase MAPK, indicating that the MAPK pathway plays a major role in the production of various pro-inflammatory mediators. Therefore, in this invention, the AMPK / MTOR pathway and MAPK pathway are selected to illustrate the anti-inflammatory mechanism of this compound composition. From the above results, it can be seen that this composition may play an autophagy regulatory role by activating AMPK and thus inhibiting MTOR phosphorylation, and affect the expression of inflammatory proteins and the release of inflammatory factors by inhibiting the phosphorylation of the MAPK pathway.

[0141] Example 8

[0142] To ensure the safety of the Tibetan medicine compound in this invention during clinical use, in this example, the maximum dose method is used to administer the maximum dose of the drug to SD male and female rats once by gavage with the drug extract powder prepared by the method of Example 2, and observe the acute toxicity reaction after administration, record information such as the toxicity reaction time and symptoms, target organs, death time, recovery time, changes in animal body weight, and death conditions, etc., to provide a basis for determining the safety range and toxicity characteristics of this test drug.

[0143] 8.1 Animal grouping

[0144] In this example, after the 14-day quarantine period, the animals are weighed on the last day of the quarantine period and randomly divided into 4 groups according to body weight stratification. There are two groups of SD male and female rats each, divided into a control group and a drug administration group; among them, there are 12 rats in the control group, 6 males and 6 females; there are 21 rats in the drug administration group, 10 females and 11 males.

[0145] 8.2 Toxicity Experiment Conducted

[0146] Based on the dissolution situation when preparing the medicated liquid and the ease of sucking the medicated liquid with a No. 16 gavage needle, the maximum administration concentration of the extract powder of the Tibetan medicine composition of the present invention is 1.5 g / ml. Since the maximum single administration volume is 20 ml / kg, the maximum single administration dose is set at 30 g / kg, which is equivalent to 210 g of crude drug / kg in terms of crude drug amount; the administration route is determined as gavage administration;

[0147] This experiment adopts the maximum single administration dose method. The administration group is given a single dose within one day, and the total dose is 30 g / kg (292 times the clinical effective dose). The negative control group is given an equal volume of distilled water. After administration, the animals are continuously observed for two weeks, and the results are determined by anatomical sampling. During the anatomical process, one male rat in the administration group died due to anesthesia, and no blood or viscera were taken, but no lesions were observed in the viscera by naked eyes; blood collection was not successful for one female rat in the administration group, and the viscera were taken, and no lesions were observed in the viscera by naked eyes.

[0148] 8.3 Experimental Indicators

[0149] (1) Body weight detection

[0150] On the day of administration and on the 1st, 2nd, 3rd, 5th, 7th, 10th, and 14th days after administration, the body weights of the animals are measured respectively.

[0151] (2) Blood biochemistry indicators

[0152] On the 14th day after administration, blood is taken from the abdominal aorta of the rats, and various biochemical indicators in the blood are detected. The indicators include AST / ALT (aspartate aminotransferase / alanine aminotransferase), BUN (blood urea nitrogen), TBI (total bilirubin), and TP (total protein), which can detect the health conditions of the liver, kidney, gallbladder, etc. and the overall nutritional health status of the body.

[0153] (3) Blood physiology indicators

[0154] On the 14th day after administration, blood is taken from the abdominal aorta of the rats, and the white blood cell count, red blood cell count, hemoglobin concentration, and platelet count in the blood are statistically analyzed using an automatic blood analyzer. It can detect the inflammatory condition, hematopoietic function, oxygen-carrying capacity, and coagulation function in the body. The overall health condition of the rats is comprehensively measured.

[0155] (4) Important organ indices

[0156] On the 14th day after administration, important organs such as the heart, liver, spleen, lungs, kidneys, and brain are taken, weighed, and the organ indices are measured. During the anatomical process, it is also observed whether there are obvious organ lesions to judge whether there is a risk of organ damage when taking a large amount of the Tibetan medicine compound of the present invention.

[0157] 8.4 Experimental Results

[0158] (1) Body weight growth rate

[0159] On the first day after administration, the body weight of female rats decreased significantly. On the second day, the body weight began to increase again. On the third day, the growth rate was slower than that of the control group. From the fourth day on, there was no significant difference in the body weight data compared with the control group. On the first day after administration, the body weight of male rats decreased extremely significantly. On the second day, it gradually returned to normal and there was no significant difference from the control group. The specific data are shown in Tables 10 and 11. From the above situation, after a single administration of the extract powder of the Tibetan medicine composition to female and male rats at the maximum dosage, the body weight growth rate index was normal.

[0160] Table 10 Body weight growth rate of female rats

[0161]

[0162] Note: Compared with the control group, ** indicates P < 0.01, and * indicates P < 0.05

[0163] Table 11 Body weight growth rate of male rats

[0164]

[0165] Note: Compared with the control group, ** indicates P < 0.01, and * indicates P < 0.05

[0166] (2) Blood biochemistry indexes

[0167] The total protein TP index is related to the nutritional requirements and material metabolism ability of animals of different ages and genders. Compared with the control group, although the total protein TP index of the male rat administration group was significantly lower, the mean values were not very different and were within the normal range according to the literature. There were no significant differences in other indexes. There were no significant differences in the blood biochemistry indexes of female rats. The specific results are shown in Tables 12 and 13. From the above situation, after a single administration of the extract powder of the Tibetan medicine composition to female and male rats at the maximum dosage, the blood biochemistry indexes were normal.

[0168] Table 12 Biochemical indexes of female rats

[0169]

[0170] Table 13 Biochemical indexes of male rats

[0171]

[0172] Note: Compared with the control group, ** indicates P < 0.01, and * indicates P < 0.05

[0173] (3) Blood physiology indexes

[0174] Compared with the control group, there were no significant differences in the blood routine values of the drug-administered groups of female and male rats, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT). The specific results are shown in Tables 14 and 15. From the above situation, it can be seen that after a single administration of the extract powder of the Tibetan medicine composition to female and male rats at the maximum dose, the blood physiological indexes were normal.

[0175] Table 14 Blood Routine Value Indexes of Female Rats

[0176]

[0177] Table 15 Blood Routine Value Indexes of Male Rats

[0178]

[0179] (4) Indexes of Important Organs

[0180] There were no significant differences in the organ indexes of the drug-administered groups of female and male rats compared with the control group. The specific results are shown in Tables 16 and 17. From the above situation, it can be seen that after a single administration of the extract powder of the Tibetan medicine composition to female and male rats at the maximum dose, the organ index indexes were normal.

[0181] Table 16 Organ Index Indexes of Female Rats (mg / g)

[0182]

[0183] Table 17 Organ Index Indexes of Male Rats (mg / g)

[0184]

[0185] 8.5 Summary

[0186] In this invention, the systemic and organ toxicity effects of the Tibetan medicine compound of this invention were evaluated by indexes such as the body weight growth rate of rats, the indexes of important organs, hematology, and blood biochemistry. The results showed that after a single large-dose intragastric administration of the extract suspension of the Tibetan medicine composition, there was a transient and slight slowdown in the body weight growth of female and male rats, which gradually returned to normal after one day. However, the indexes of organs such as the heart, liver, spleen, lung, kidney, and brain, hematology, and blood biochemistry showed no obvious abnormalities, and no clear tissue damage was found during pathological anatomy. This indicates that the maximum single dose of the Tibetan medicine composition of this invention is 30 g / kg, and no obvious toxic symptoms were observed during the entire test period. This dose is equivalent to 292 times the clinically proposed dose.

Claims

1. A Tibetan medicine composition for treating chronic obstructive pneumonia, characterized in that the composition is made from the following raw medicinal materials in parts by weight: 120 - 160 parts of gentian flower, 50 - 55 parts of ash bark, 50 - 55 parts of lagotis glauca, 19 - 25 parts of nutmeg, 50 - 60 parts of travertine, 20 - 30 parts of zha xun paste, 75 - 85 parts of liquorice.

2. The Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 1, characterized in that the composition is made from the following raw medicinal materials in parts by weight: 140 - 160 parts of gentian flower, 50 - 55 parts of ash bark, 50 - 55 parts of lagotis glauca, 19 - 23 parts of nutmeg, 50 - 55 parts of travertine, 26 - 30 parts of zha xun paste, 75 - 79 parts of liquorice.

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

4. The Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 3, characterized in that the preparation is one of granules, tablets, and capsules.

5. A preparation method of the Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 1 or 2, characterized in that the method comprises the following steps: taking gentian flower, ash bark, lagotis glauca, nutmeg, and liquorice medicinal materials in proportion, soaking in water and then decocting or heating under reflux for extraction, filtering, concentrating the filtrate to obtain a concentrated extract, adding zha xun paste and mixing, then performing alcohol precipitation, taking the supernatant and concentrating, then combining with travertine, drying, pulverizing, and sieving to obtain an extract powder.

6. The preparation method of the Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 5, characterized in that the soaking time in water is 30 - 60 min.

7. The preparation method of the Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 5, characterized in that the decocting or heating under reflux extraction is performed 2 - 3 times, each decocting or extraction is 1 - 2 hours, and the water consumption for each decocting or extraction is 8 - 14 times the weight of the medicinal materials.

8. The preparation method of the Tibetan medicine composition for treating chronic obstructive pneumonia according to claim 5, characterized in that the alcohol precipitation is adding 3 - 4 times the volume of 95% ethanol, and the alcohol precipitation time is 24 hours.

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

Images