Traditional Chinese medicine composition for treating cough variant asthma and application thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating cough variant asthma and application thereof, and belongs to the field of traditional Chinese medicine preparations. The traditional Chinese medicine composition is prepared from radix bupleuri, herba ephedrae, herba schizonepetae, semen armeniacae amarae, radix platycodonis, fructus aurantii immaturus, exocarpium citri grandis, rhizoma phragmitis, rhizoma pinelliae, rhizoma belamcandae, the traditional Chinese medicine composition can obviously improve the condition of weight loss of guinea pigs after combined molding of ovalbumin and capsaicin, reduces the cough frequency and the lung focus area, effectively inhibits inflammatory factors, and can reach the treatment level equivalent to that of montelukast sodium.

Description

Technical Field

[0001] The present invention provides a traditional Chinese medicine composition for treating cough variant asthma and its application, belonging to the field of traditional Chinese medicine preparations. Background Art

[0002] Cough variant asthma (CVA) is a respiratory disease with repeated cough as the main or only clinical manifestation. Its incidence rate ranks first among adult chronic coughs and it belongs to refractory chronic respiratory diseases in clinical practice. Pathologically, eosinophilic granulocyte inflammation can be detected in the peripheral blood of CVA patients, and the CT results of some patients show diffuse ground-glass opacities in the lungs. Clinically, the treatment drugs for CVA mainly include antibiotics, antihistamines, glucocorticoids, cough suppressants, etc. These drugs target the processes such as the release of lipid inflammatory mediators and eosinophil activation in the pathogenesis of CVA. Although they have certain effects, most drugs have obvious adverse reactions and are accompanied by withdrawal symptoms of disease recurrence.

[0003] In "Ren Shen Bai Du San" in "Taiping Huimin He Ji Ju Fang", it is recorded that: Bupleurum chinense (two qian), Ligusticum wallichii (one and a half qian), Peucedanum praeruptorum, Glycyrrhiza uralensis, Panax ginseng, Platycodon grandiflorum, Notopterygium incisum, Angelica pubescens, Poria cocos, Aurantii Fructus Immaturus, each one qian, a little Mentha haplocalyx Briq. It is used for exogenous cough, stubborn cough, and chronic cough, and is currently commonly used to treat childhood cough variant asthma and persistent cough after pneumonia. "When affected by cold, one gets sick. Slightly, it is cough; severely, it is diarrhea or pain." Cough variant asthma is caused by slightly cold pathogens lingering in the lungs, and the condition lingers and does not heal. It is treated with warm and moist Xinjin.

[0004] Chinese Patent CN107737285A discloses a decoction drug for treating childhood cough and its preparation method. Its prescription is: Perilla seed 5 - 12g, Mori Folium 5 - 12g, Forsythia suspensa 15 - 28g, Chrysanthemi Flos 5 - 10g, Platycodon grandiflorum 3 - 10g, Ephedra sinica 3 - 12g, Armeniacae Semen Amarum (peeled and pointed) 5 - 9g, Imperatae Rhizoma 9 - 35g, Phragmitis Rhizoma 15 - 35g, Pueraria lobata 8 - 16g, Citri Reticulatae Pericarpium 3 - 10g, Lonicera japonica 10 - 25g, Thunbergii Fritillaria Bulb 3 - 12g, Isatidis Folium 9 - 16g, Peucedanum praeruptorum 3 - 10g, Pinelliae Rhizoma Praeparatum 3 - 10g, Aster tataricus 3 - 16g, Belamcandae Rhizoma 3 - 12g, Tussilaginis Flos 3 - 10g, Citri Grandis Exocarpium 3 - 7g, Poria cocos 9 - 16g, Lilium brownii 6 - 15g, Ophiopogonis Radix 6 - 15g, Glehniae Radix 15 - 30g, Aurantii Fructus Immaturus (fried) 3 - 10g, Polygonati Odorati Rhizoma 6 - 15g, Stemona japonica 3 - 10g, Scutellaria baicalensis 3 - 10g, Glycyrrhiza uralensis 3 - 12g. However, its treatment is for childhood cough patients and does not conduct research on cough variant asthma.

[0005] Chinese Patent CN112569322A discloses a pharmaceutical composition with antitussive and antiasthmatic effects. Its prescription is as follows: 0.7 - 1.4 parts of honey-fried Ephedra, 0.7 - 1.4 parts of Bitter Apricot Seeds, 1.4 - 2.8 parts of Gypsum, 0.7 - 1.4 parts of Perilla Seeds, 0.7 - 1.4 parts of Peucedanum praeruptorum Dunn, 0.7 - 1.4 parts of Schizonepeta tenuifolia Briq., 0.7 - 1.4 parts of Dried Tangerine Peel, 0.7 - 1.4 parts of Platycodon grandiflorum, 0.7 - 1.4 parts of Belamcanda chinensis, 1.4 - 2.8 parts of Loquat Leaves, 0.9 - 1.8 parts of Stemona japonica, 0.9 - 1.8 parts of Inula britannica L., 0.7 - 1.4 parts of Licorice Root. It has the effects of dispersing lung qi to arrest cough, clearing heat and relieving asthma. However, it does not conduct research on cough variant asthma.

[0006] Zhao Lisha et al. in the clinical efficacy of Xiaoqinglong Decoction combined with Sanzi Yangqin Decoction in the treatment of cough variant asthma in children (DOI: 10.16458 / i.cnki.1007 - 0893.2023.03.011), on the basis of nebulized inhalation of budesonide and taking montelukast sodium, gave Xiaoqinglong Decoction combined with Sanzi Yangqin Decoction. The formula is as follows: 5g each of fried Ephedra, white peony root, poria cocos, bitter apricot seeds, and earthworm, 12g of cinnamon twig, 3g each of asarum and dried ginger, 9g each of prepared pinellia and roasted licorice root, 6g each of schisandra chinensis and dried tangerine peel, 15g of perilla seeds, 10g each of white mustard seeds, radish seeds, magnolia officinalis, peucedanum praeruptorum dunn, mulberry bark, and inula britannica l., 3g of chuanxiong rhizome. Its total effective rate is 42%, which is higher than that of the pure western medicine group. However, the effect of single treatment is unknown and the treatment effect needs to be improved. Summary of the Invention

[0007] In order to solve the deficiencies existing in the prior art, the present invention provides a traditional Chinese medicine composition for treating cough variant asthma and its application. The traditional Chinese medicine composition of the present invention can significantly improve the weight loss of guinea pigs after combined modeling with ovalbumin and capsaicin, reduce the number of coughs and the area of lung lesions, effectively inhibit inflammatory factors, and can achieve a treatment level equivalent to that of montelukast sodium.

[0008] To achieve the above object, the technical scheme adopted by the present invention is as follows:

[0009] On the one hand, the present invention provides a traditional Chinese medicine composition for treating cough variant asthma, including bupleurum root, ephedra, schizonepeta tenuifolia briq., bitter apricot seeds, platycodon grandiflorum, immature bitter orange, tangerine peel, reed rhizome, pinellia ternata, belamcanda chinensis, white stiff silkworm, pogostemon cablin, and mentha haplocalyx briq.

[0010] Furthermore, the mass ratio of the bupleurum root, ephedra, and schizonepeta tenuifolia briq. is: (4 - 6):(2 - 4):(1 - 3).

[0011] Preferably, the mass ratio of the bupleurum root, ephedra, and schizonepeta tenuifolia briq. is: (5 - 6):(3 - 4):(2 - 3).

[0012] Furthermore, the mass ratio of bitter apricot kernels, immature bitter orange, and pinellia tuber is: (2 - 4):(1 - 3):(1 - 2).

[0013] Preferably, the mass ratio of bitter apricot kernels, immature bitter orange, and pinellia tuber is: (3 - 4):(2 - 3):2.

[0014] Furthermore, the traditional Chinese medicine composition, in parts by weight, comprises 4 - 6 parts of bupleurum root, 2 - 4 parts of honey - fried ephedra, 1 - 3 parts of schizonepeta spike, 2 - 4 parts of bitter apricot kernels, 1 - 3 parts of platycodon root, 1 - 3 parts of immature bitter orange, 1 - 2 parts of tangerine peel, 1 - 3 parts of phragmites rhizome, 1 - 2 parts of pinellia tuber, 3 - 5 parts of belamcanda root, 1 - 3 parts of white muscardine silkworm, 1 part of pogostemon cablin, and 1 part of mentha haplocalyx Briq.

[0015] Preferably, the traditional Chinese medicine composition, in parts by weight, comprises 6 parts of bupleurum root, 4 parts of honey - fried ephedra, 3 parts of schizonepeta spike, 4 parts of bitter apricot kernels, 3 parts of platycodon root, 3 parts of immature bitter orange, 2 parts of tangerine peel, 3 parts of phragmites rhizome, 2 parts of pinellia tuber, 5 parts of belamcanda root, 2 parts of white muscardine silkworm, 1 part of pogostemon cablin, and 1 part of mentha haplocalyx Briq.

[0016] In a second aspect, the present invention provides the use of the traditional Chinese medicine composition in the preparation of a medicament for treating respiratory diseases.

[0017] Furthermore, the respiratory disease is cough - variant asthma.

[0018] In a third aspect, the present invention provides a medicament for treating respiratory diseases, comprising the traditional Chinese medicine composition.

[0019] The beneficial effects of the present invention are as follows:

[0020] In the present invention, bupleurum root and ephedra are used as the monarch drugs, schizonepeta spike, bitter apricot kernels, platycodon root, and immature bitter orange are used as the ministerial drugs, tangerine peel, pinellia tuber, belamcanda root, phragmites rhizome, and white muscardine silkworm are used as the adjuvant drugs, and pogostemon cablin and mentha haplocalyx Briq are used as the guiding drugs. With the specific dosage relationship of bupleurum root, ephedra, and schizonepeta spike and the dosage relationship of bitter apricot kernels, immature bitter orange, and pinellia tuber, the traditional Chinese medicine composition of the present invention can significantly improve the weight loss of guinea pigs after the combined modeling with ovalbumin and capsaicin, reduce the cough frequency and the area of lung lesions, effectively inhibit inflammatory factors, and can reach a treatment level equivalent to montelukast sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a graph of the body weight change of CVA guinea pigs.

[0022] Figure 2 It is a graph of the cough frequency of CVA guinea pigs.

[0023] Figure 3 It is a pathological structure diagram of the lung tissue of CVA guinea pigs, scale bar 200μm.

[0024] Figure 4It is the pathological structure diagram of the trachea of CVA guinea pigs, with a scale of 200μm.

[0025] Figure 5 It is the lung imaging diagram of CVA guinea pigs.

[0026] Figure 6 It is the Micro CT lung injury score diagram of the lungs of CVA guinea pigs (n = 8).

[0027] Figure 7 It is the Masson staining diagram of the pathological morphology of the lung tissue of CVA guinea pigs.

[0028] Figure 8 It is the Masson staining diagram of the pathological morphology of the trachea tissue of CVA guinea pigs.

[0029] Figure 9 It is the mucin diagram in the trachea tissue of CVA guinea pigs.

[0030] Figure 10 It is the mucin diagram in the lung tissue of CVA guinea pigs.

[0031] Figure 11 It is the immune cell subset diagram in the peripheral blood of CVA guinea pigs (n = 8).

[0032] Figure 12 It is the immune cell subset diagram in the alveolar lavage fluid of CVA guinea pigs (n = 8).

[0033] Figure 13 It is the inflammatory factor mRNA expression diagram in the lung and trachea tissues of CVA guinea pigs (n = 8).

[0034] Figure 14 It is the inflammatory factor level diagram in the serum and alveolar lavage fluid of CVA guinea pigs (n = 8).

[0035] Figure 15 It is the inflammatory factor mRNA expression diagram in the lung, trachea, and dorsal root ganglion tissues of CVA guinea pigs (n = 8).

[0036] Figure 16 It is the neurogenic inflammatory factor level diagram in the serum and alveolar lavage fluid of CVA guinea pigs (n = 8).

[0037] Figures 1 - 16In the following: control is the blank group, Model is the model group, MS is the montelukast sodium group, SJS-L is the low-dose Shujin San group, SJS-M is the medium-dose Shujin San group, and SJS-H is the high-dose Shujin San group; # indicates p < 0.05 compared with the blank group, ## indicates p < 0.01 compared with the blank group, indicates p < 0.005 compared with the blank group; * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, *** indicates p < 0.005 compared with the model group.

[0038] Figure 5 For the control diagram, Model diagram, MS diagram, SJS-L diagram, SJS-M diagram, and SJS-H diagram, the black background parts in the upper right corner are the numbers, time, and parameters automatically generated by the system and cannot be modified, which do not affect the technical effects of the present invention. Detailed implementation manners

[0039] In order to make the technical means, creative features, achieved purposes, and functions of the present invention easy to understand, the following specific embodiments are used to further clarify the present invention. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitations are imposed on their sources. The technical and scientific terms used in the embodiments have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0040] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in the technical field.

[0041] Table 1 shows the main experimental reagents and instruments of the present invention.

[0042] Table 1

[0043]

[0044]

[0045] I. Embodiments and comparative examples

[0046] Example 1 A traditional Chinese medicine composition for treating cough variant asthma

[0047] Bupleurum chinense 6 parts, honey-fried Ephedra 4 parts, Schizonepeta tenuifolia 3 parts, bitter apricot kernel 4 parts, Platycodon grandiflorum 3 parts, Fructus Aurantii Immaturus 3 parts, Citrus reticulata Blanco cv. Tomentosa 2 parts, Phragmites communis Trin. 3 parts, Pinellia ternata 2 parts, Belamcanda chinensis 5 parts, Bombyx batryticatus 2 parts, Herba Pogostemonis 1 part, Mentha haplocalyx Briq. 1 part.

[0048] Preparation method:

[0049] The raw materials are ground and passed through a 100-mesh sieve to obtain a powder. The powder is extracted with 75% ethanol, concentrated to obtain an extract, and then freeze-dried to obtain the product.

[0050] II. Animal experiments

[0051] 1. Experimental drugs

[0052] Capsaicin was purchased from Chengdu Efa Biotechnology Co., Ltd. (CAS No. 404-86-4; molecular formula C 18 H 27 NO 3 ) and its chemical structure is:

[0053] Example 1 of the present invention (hereinafter referred to as Shujin Powder) was provided by the Modern Traditional Chinese Medicine Innovation Center of Tianjin University of Traditional Chinese Medicine. The clinical dosage of Shujin Powder is 117 g / day, and the extract yield is 20% (extract yield = mass of freeze-dried powder / mass of raw medicinal materials

[0054] ×100). That is, the clinical dose of Shujin Powder is 117 g×0.20 / 60 kg (the adult body weight is calculated as 60 kg), and the conversion coefficient between humans and guinea pigs is 4.625. That is, the equivalent dose of guinea pigs and humans for clinical drug administration = 117 g*0.20 / 60 kg×4.625 = 1.8 g / kg. The experiment adopted 1, 3, and 6 times the clinical dose, that is, 1.8 g / kg, 5.4 g / kg, and 10.8 g / kg.

[0055] Montelukast sodium was purchased from Shijiazhuang Pharmaceutical Group Ouyi Pharmaceutical Co., Ltd. (CAS No. 151767-02-1; molecular formula C 35 H 35 ClNO 3 S·Na) and its chemical structure is: The present invention adopts the clinical equivalent dose, 0.0015 g / kg

[0056] 2. Experimental animals

[0057] Forty-eight SPF-grade male Hartley guinea pigs, weighing 220-250 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The certificate number is: 110011241104485713, and the license number is: SCXK2021-0011. This experimental procedure was approved by the Animal Research Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2024062z1625) and was raised in the Animal Center of Tianjin University of Traditional Chinese Medicine.

[0058] 3. Experimental methods

[0059] (1) Preparation of solutions and drugs

[0060] Ransom Powder: Prepare it for immediate use. Weigh 5.4 g, 16.2 g, and 32.4 g of freeze-dried powder of Ransom Powder extract, add 30 mL of pure water respectively to prepare medicinal solutions with concentrations of 0.18 g / mL, 0.54 g / mL, and 1.08 g / mL. Mix well, ultrasonicate for 3 h, store at 4°C, and administer by gavage at a dose of 1.0 mL / 100 g once a day.

[0061] Montelukast Sodium: Prepare it for immediate use. Weigh 10 mg of montelukast sodium tablets, add 66.6 mL of pure water to prepare a medicinal solution with a concentration of 0.15 mg / mL. Mix well by ultrasonication, store at 4°C, and administer by gavage at a dose of 1.0 mL / 100 g once a day.

[0062] 4% Ovalbumin Solution: Prepare it for immediate use. Dissolve 1.5 g of ovalbumin powder in 37.5 mL of normal saline to prepare a 4% ovalbumin solution. Mix well before use. The modeling dose is 0.5 mL / animal.

[0063] 1% Ovalbumin Solution: Prepare it for immediate use. Dissolve 0.05 g of ovalbumin powder in 5 mL of normal saline to prepare a 1% ovalbumin solution. Mix well before use.

[0064] 2% Aluminum Hydroxide Solution: Prepare it for immediate use. Dissolve 0.3 g of aluminum hydroxide powder in 15 mL of normal saline to prepare a 2% aluminum hydroxide solution. Mix well before use. The modeling dose is 0.2 mL / animal.

[0065] Capsaicin Solution: Prepare it for immediate use. Weigh 1.527 mg of capsaicin powder, dissolve it in 8 mL of normal saline, add 1 mL of Tween-80 and 1 mL of absolute ethanol to prepare a 0.5 mM capsaicin solution. Mix well before use.

[0066] (2) Establishment of cough variant asthma model guinea pigs and administration

[0067] The CVA model guinea pigs were replicated by using ovalbumin and aluminum hydroxide to stimulate sensitization and capsaicin to stimulate. The animals were randomly divided into a blank group (Control), a model group (Model), a low-dose Shujin powder group (SJS-L), a medium-dose Shujin powder group (SJS-M), a high-dose Shujin powder group (SJS-H), and a montelukast sodium group (MS), with 8 animals in each group. Except for the blank group, on the 1st and 8th days, the remaining groups were intramuscularly injected with 0.5 mL of 4% ovalbumin solution and intraperitoneally injected with 0.2 mL of 2% aluminum hydroxide solution for sensitization. Since the 9th day, the guinea pigs in the model group, the low-dose Shujin powder group, the medium-dose Shujin powder group, the high-dose Shujin powder group, and the montelukast sodium group were placed in a closed atomization exposure chamber and ultrasonically atomized and stimulated with 1% ovalbumin solution for 20 min once a day for 7 days. The blank group was only intramuscularly injected with 0.5 mL of normal saline and intraperitoneally injected with 0.2 mL of normal saline on the 1st and 8th days, and ultrasonically atomized with normal saline alone for 20 min from the 9th to 15th days.

[0068] When performing the capsaicin stimulation experiment, guinea pigs were stimulated by aerosol inhalation of 0.5 mM capsaicin solution. When morphological changes such as rapid breathing, abdominal muscle contraction, and coughing appeared in the guinea pigs, the number of coughs was counted within 3 min. Coughing > 10 times indicated successful model establishment.

[0069] After successful model establishment, the low-dose Shujin powder group, the medium-dose Shujin powder group, and the high-dose Shujin powder group began to intragastrically administer the Shujin powder extract solution at doses of 1.8 g / kg, 5.4 g / kg, and 10.8 g / kg, respectively. The montelukast sodium group intragastrically administered the montelukast sodium solution at a dose of 1.5 mg / kg, and the administration continued for 7 days. The blank group and the model group intragastrically administered the corresponding dose of normal saline. Index detection and sample collection were performed on the 23rd day.

[0070] III. Detection methods and results

[0071] 1. Shujin powder slows down the weight loss rate of CVA guinea pigs and reduces the number of coughs

[0072] (1) Detection of body weight and number of coughs

[0073] From the first day of model establishment, the weight of each guinea pig was weighed at a fixed time every day until the day before sample collection. After successful model establishment and after the animals in each group were intragastrically administered the corresponding drugs, the number of coughs was detected every other day. The guinea pigs were placed in an animal atomization chamber and atomized and inhaled 0.5 mmol / L capsaicin solution for 1 min, and then the guinea pigs were immediately taken out and the number of coughs within 3 min was counted.

[0074] (2) Experimental results

[0075] As Figure 1As shown, after the guinea pigs were modeled by aerosol inhalation of OVA combined with capsaicin, their body weights decreased. After drug administration intervention, the body weights of the drug administration groups recovered rapidly compared with the model group, and the body weights of the montelukast sodium group and the high-dose Shujin San group recovered faster than those of the medium-dose and low-dose Shujin San groups.

[0076] The number of coughs, as the most intuitive manifestation reflecting the cough sensitivity of CVA guinea pigs, the experimental results are as Figure 2 , and the low, medium, and high doses of Shujin San can significantly reduce the number of coughs in CVA guinea pigs.

[0077] 2. Shujin San alleviates the lung and tracheal lesions and mucin secretion in CVA guinea pigs

[0078] (1) HE staining, Masson staining and Micro CT detection

[0079] HE staining: The guinea pigs were sacrificed and the chest cavity was opened to fully expose the lung tissue. The upper and middle lobes of the right lung of the guinea pigs and 0.5 cm of the trachea near the lung were immediately placed in 10% formalin solution and fixed for more than 24 h. After dehydration with gradient concentration ethanol, it was embedded in paraffin and sectioned. Then the tissue sections were deparaffinized with xylene, rehydrated with ethanol of different concentration gradients, and stained with hematoxylin staining solution for 5 min. The floating color was washed off with distilled water; differentiated with the differentiating solution for 3 min, rinsed with tap water 2 times, 2 min each time; eosin staining solution for 30 s, slightly rinsed with distilled water. Dehydrated and sealed, sealed with neutral gum, and observed under the microscope.

[0080] Masson staining: The sections were placed in an incubator at 60 °C for 60 min; the sections were deparaffinized to water with xylene Ⅰ, Ⅱ and gradient ethanol; the Weigert iron hematoxylin staining solution was prepared before use and dropped to cover the sections for staining for 10 min; the excess staining solution was washed off with distilled water, and the acidic differentiating solution was dropped for differentiation for 15 s, and rinsed with distilled water for 30 s; the Masson bluing solution was dropped for bluing for 5 min, and rinsed with distilled water for 30 s; the ponceau fuchsin staining solution was dropped for staining for 5 min; the weak acid working solution was dropped for washing for 30 s; the excess liquid was poured off, the phosphomolybdic acid solution was dropped for treatment for 2 min, and the weak acid working solution was dropped for washing for 30 s; the excess liquid was poured off, the aniline blue staining solution was dropped for staining for 2 min, and the weak acid working solution was dropped for washing for 30 s; dehydrated and sealed, sealed with neutral gum, and observed under the microscope.

[0081] Alcian blue - periodic acid - Schiff staining (AB - PAS): Place the sections in an incubator at 60 °C for 60 min; Place the sections in xylene I and II, and dewax them to water through gradient ethanol; Stain with Alcian blue staining solution for 4 min, wash with tap water 3 times, 2 min each time; Oxidize in the oxidant for 5 min. Rinse with tap water for 1 min, soak in distilled water 2 times, 15 s each time; Stain with Schiff staining solution for 8 min, and rinse with water for 8 min; Stain the nuclei with hematoxylin staining solution for 1 min, and wash with water; Differentiate with acidic differentiating solution for 2 s, and wash with water; Blue with Scott bluing solution for 2 min, and wash with water for 2 min; Dehydrate and mount the slides, seal with neutral balsam, and observe under the microscope.

[0082] Micro CT detection: Observe Micro CT one day before sampling. After anesthetizing the guinea pigs with tribromoethanol, fix them horizontally on the animal support matching the Micro CT. Adjust the distance and position to make the lung tissue of the guinea pigs directly below the square frame. Set the instrument parameters, with a scanning resolution of 40 mm, an exposure time of 2000 ms, a voltage of 90 kV, a current of 180 μA, and a 360° full - range scan. The graphic resolution is 40 mm. First, quickly scan and observe, and then set the scanning time to 4.5 min. Subsequently, further conduct imaging scoring. For those without lung consolidation, the score is 0; <5%, the score is 1; 5% - 25%, the score is 2; 25% - 50%, the score is 3; 50% - 75%, the score is 4; >75%, the score is 5.

[0083] (2) Experimental results

[0084] Detect the pathological status of the lungs and trachea of CVA guinea pigs by HE staining, as Figure 3 shown. Compared with the Control group, after nebulized inhalation of OVA and capsaicin for modeling, the alveolar septum of guinea pigs thickened, there was obvious infiltration of inflammatory cells, the capillaries and veins in the alveolar wall dilated, and there were obvious bleeding points in the lungs; Administering different doses of Shujinsan could improve the thickening of the alveolar septum in CVA guinea pigs, reduce leukocyte infiltration, and improve the bleeding of capillaries and veins in the alveolar wall.

[0085] Meanwhile, the tracheal lesions of guinea pigs in the CVA group were also visible. As Figure 4 shown, the tracheal mucosal epithelium in the Control group was continuous and intact, there were basically no exfoliated cells in the lumen, and there was basically no infiltration of inflammatory cells in the wall and surrounding tissues. Compared with the Control group, after nebulized inhalation of OVA and capsaicin for modeling, the adhesion of the tracheal mucosal epithelium in guinea pigs was poor and exfoliated, there was a small amount of infiltration of inflammatory cells in the wall and surrounding tissues, small blood vessel hyperplasia, the columnar cells were flattened, and the cilia were arranged irregularly; After administering different doses of Shujinsan, the degree of exfoliation of the tracheal mucosal epithelium and the degree of infiltration of inflammatory cells in the wall and surrounding tissues were reduced to varying degrees compared with the model group.

[0086] As Figure 5As shown, the bilateral lung parenchyma density of the guinea pigs in the Control group was uniform, the lung texture was clear, and the texture formed by obvious bronchi and blood vessels showed natural running; compared with the Control group, after nebulized inhalation of OVA and capsaicin for modeling, there were patchy density-increased shadows in the bilateral lungs of the guinea pigs, the lung texture was blurred, and round or oval density-increased shadows appeared around the hilum of the lung, and the lesion site was significantly enlarged; compared with the model group, in the groups of Shujinsan with different doses, the lesion area of the guinea pigs' lungs decreased, the lung texture tended to be clear, and the patchy density-increased shadows in the bilateral lungs decreased.

[0087] The results of lung imaging scoring showed that compared with the Control group, the lesion site in the model group increased significantly; the lesion area of the guinea pigs' lungs in the groups with different doses decreased significantly ( Figure 6 ).

[0088] As Figure 7 shown, the results of Masson staining showed that the lung tissue structure of the guinea pigs in the Control group was basically normal, with a small amount of blue collagen fiber deposition. Compared with the Control group, the lung tissue structure in the model group was significantly damaged, and a large amount of blue collagen fiber deposition was visible, especially around the bronchial wall and blood vessel wall. After administration of Shujinsan with different doses, the lung tissue structure of the guinea pigs was partially damaged and the collagen fiber deposition decreased.

[0089] Since the above phenomenon was particularly obvious around the bronchial wall, the tracheal tissue was further selected for Masson staining. As Figure 8 shown, compared with the Control group, collagen fiber deposition appeared in the submucosa of the model group, and the collagen fiber deposition decreased after administration of Shujinsan with different doses.

[0090] The results of AB-PAS were as Figure 9 shown. Compared with the guinea pigs in the Control group, the expression of epithelial mucin in the tracheal tissue of the guinea pigs in the model group increased significantly, and the increase in the expression of epithelial mucin in the tracheal tissue of the guinea pigs in the groups of Shujinsan with different doses was inhibited. The same phenomenon was shown in the lung tissue. As Figure 10 shown, compared with the guinea pigs in the Control group, the expression of mucin increased in the epithelium and around the bronchi of the lung tissue of the guinea pigs in the model group, and the expression of its mucin decreased after administration of Shujinsan with different doses.

[0091] 3. Shujinsan improves the abnormal changes of immune cell subsets in CVA guinea pigs

[0092] (1) Blood routine detection and bronchoalveolar lavage fluid detection

[0093] Blood routine test: After collecting blood from the abdominal aorta of guinea pigs, 20 μL of whole blood was placed in an anticoagulant tube containing 1.5 mL of sodium citrate, and a fully automatic blood cell analyzer was used for blood routine test. The test items included white blood cells (WBC), eosinophils (Eos), basophils (Bas), neutrophils (Neu), lymphocytes (Lym), and monocytes (Mon).

[0094] Alveolar lavage fluid test: After blood collection, the chest cavity of guinea pigs was exposed, the right 3-lobe lung tissue was gently stirred with a cotton swab and then ligated with silk thread. 1 mL of PBS was aspirated in advance with a syringe. The lavage needle was inserted through the tracheal opening and ligated with silk thread to prevent air leakage during lavage. After fixing the lavage needle to prevent air leakage, it was slowly injected into the left lung, and then aspirated back for lavage. This was repeated 3 - 5 times to obtain the lavage fluid, and the recovery rate was about 80% - 90%.

[0095] ① Determination of the total protein concentration in the supernatant of alveolar lavage fluid

[0096] The supernatant obtained by centrifuging at 2700 rpm for 10 min was transferred to a new centrifuge tube for determination of the total protein concentration of BALF, and the BCA method was used for determination. First, prepare the BSA standard solution. According to the need, the standard BSA solution with a concentration of 5 mg / mL was serially diluted with PBS to BSA standards of 2000, 1600, 1200, 800, 400, 200, 100, 50, 0 μg / mL.

[0097] The standard solution and the sample solution were added to the corresponding wells of the 96-well plate in turn (20 μL / well). The pre-prepared BCA reagent (A liquid: B liquid = 50:1) was mixed evenly in 9 standard solution wells, and for the remaining samples, after adding the samples first, 200 μL of the working solution was immediately added, mixed well, and incubated in a 37°C incubator for 15 min to allow sufficient reaction. The OD value was detected at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader; a standard curve was plotted, an equation was listed according to the standard curve, and the OD value was substituted into the equation to calculate the total protein concentration in the alveolar lavage supernatant.

[0098] ② White blood cell classification and counting

[0099] The centrifuged cell pellet was resuspended with 100 μL of PBS, and after being blown evenly, 20 μL of the cell suspension was pipetted into a fully automatic cell counter for counting and classification.

[0100] (2) Experimental results

[0101] Increased in peripheral blood and respiratory tract secretions during asthma attacks. Such as Figure 11As shown in the figure, compared with the control group, the number of white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), neutrophils (Neu), eosinophils (Eos), and basophils (Bas) in the peripheral blood of guinea pigs in the model group increased significantly compared with the blank group. After administration of different doses of Ransom Powder, the number of white blood cells, neutrophils, eosinophils, and basophils was significantly reduced.

[0102] The same phenomenon was observed in guinea pig bronchoalveolar lavage fluid (BALF), such as Figure 12 The results showed that compared with the Control group, the number of white blood cells (WBC), lymphocytes (Lym), monocytes (Mon), neutrophils (Neu), and eosinophils (Eos) in the BALF of guinea pigs in the model group increased significantly. After administration of different doses of Ransom Powder, the number of white blood cells, neutrophils, and eosinophils was significantly reduced. At the same time, the quantitative data of BCA in bronchoalveolar lavage fluid showed that compared with the Control group, the total protein content of guinea pigs in the model group was significantly increased, and the protein content was reduced after administration of different doses of Ransom Powder, indicating that it had a significant inhibitory effect on its lung inflammation.

[0103] 4. Ransom powder reduces the levels of inflammatory factors and neurogenic inflammatory factors in CVA guinea pigs

[0104] (1) Enzyme-linked immunosorbent assay (ELISA), RNA extraction and RT-qPCR

[0105] ELISA:

[0106] Before the experiment, the serum was thawed on ice, and the contents of cytokines TNF-α, IL-6, IL-4 and neurogenic inflammatory factors CGRP and SP in guinea pig serum and bronchoalveolar lavage fluid (BALF) were detected by ELISA. The concentration gradient standard solution, biotin antigen and washing solution were prepared according to the instructions of the kit. 50 μL of the corresponding solution was added to each well and covered with a membrane, and incubated at 37°C for 30 minutes. The solution was discarded, and the washing solution was added and discarded after 30 seconds. Repeat 5 times. 50 μL of HRP was added to cover the membrane and incubated at 37°C for 30 minutes. Repeat the steps to wash the plate. 50 μL of color developer A and 50 μL of color developer B were added to each well, mixed and incubated at 37°C in the dark for 10 minutes, and 50 μL of stop solution was added to each well. The OD value was measured at a wavelength of 450 nm, a standard curve was drawn, and the sample concentration was calculated.

[0107] RNA extraction:

[0108] ① RNA extraction from lung, trachea, and dorsal root ganglion tissues

[0109] Guinea pig lung tissue was cut into 1×1 mm 3For the tissue block, add 1 mL of LB lysis buffer, break the tissue, after shaking for 4 min, add 200 μL of chloroform to every 1 mL of LB lysis buffer. Centrifuge at 4°C and 12,000 rpm for 10 min, take 400 μL of the upper aqueous phase, and mix well with 200 μL of absolute ethanol.

[0110] Cut 5 mm of guinea pig tracheal tissue, add 1 mL of LB lysis buffer, break the tissue, after shaking for 4 min, add 200 μL of chloroform to every 1 mL of LB lysis buffer. Centrifuge at 4°C and 12,000 rpm for 10 min, take 400 μL of the upper aqueous phase, and mix well with 200 μL of absolute ethanol.

[0111] Isolate 6 guinea pig dorsal root ganglion tissues, add 1 mL of LB lysis buffer, break the tissue, after shaking for 4 min, add 200 μL of chloroform to every 1 mL of LB lysis buffer. Centrifuge at 4°C and 12,000 rpm for 10 min, take 400 μL of the upper aqueous phase, and mix well with 200 μL of absolute ethanol.

[0112] Transfer the obtained sample pretreatment mixture to a RNA adsorption column, centrifuge at 12,000 rpm for 1 min, discard the solution at the bottom of the tube; then add the protein removal solution, after removing the protein, wash twice with the washing solution. Centrifuge the empty column to remove the residual washing solution, replace the new tube, add 40 μL of enzyme-free water, and let it stand for 2 min. Centrifuge at 12,000 rpm for 1 min; collect the RNA solution.

[0113] ②RNA concentration determination

[0114] Place the extracted RNA solution on ice, take 1.5 μL of the sample RNA and add it to the RNA concentration measuring instrument for concentration measurement.

[0115] Reverse transcription:

[0116] RNA is reverse transcribed to synthesize cDNA

[0117] Remove residual genomic DNA, quantify the RNA to 1000 ng, 12 μL, add 3 μL of 5× gDNADigester Mix to each sample

[0118] Prepare the reverse transcription reaction system as shown in Table 2 (20 μL system)

[0119] Table 2

[0120]

[0121]

[0122] Place it in a thermostatic mixer at 25°C for 5 minutes; 55°C for 15 minutes; 85°C for 5 minutes.

[0123] RT-qPCR:

[0124] The real-time quantitative RT-qPCR reaction system was prepared according to Table 3.

[0125] Table 3

[0126]

[0127] Reaction conditions:

[0128] 95°C, 3 min; 95°C, 30 s; 55°C, 30 s; 72°C, 30 s, for 39 cycles.

[0129] Result analysis:

[0130] Using the analysis software of the RT-PCR instrument from Bio-Rad, USA, the corresponding Ct value of each sample was obtained. Based on the Ct value, 2-ΔΔCt was calculated. With GAPDH as the internal reference gene, the relative expression level was calculated and statistical analysis was performed.

[0131] The primers involved in this experiment were purchased from Sangon Biotech (Shanghai). The primer sequences are shown in Table 4.

[0132] Table 4

[0133]

[0134]

[0135] (2) Experimental results

[0136] As Figure 13 shown, compared with the Control group, the mRNA levels of the inflammatory factor TNFα and the airway inflammatory factor IL-4 in the lung tissue and tracheal tissue of guinea pigs in the model group were significantly increased; after administration of different doses of Shujinsan, the expression levels of the inflammatory factor TNFα and the airway inflammatory factor IL-4 mRNA in the lung tissue and tracheal tissue of guinea pigs were significantly decreased.

[0137] As Figure 14 the results showed, compared with the Control group of rats, the levels of the inflammatory factors IL-6, TNF-α, and IL-4 in the serum and bronchoalveolar lavage fluid of guinea pigs in the model group were all significantly increased; compared with the model group of guinea pigs, the increase in the levels of the cytokine inflammatory factors IL-6, TNF-α, and IL-4 in the serum and bronchoalveolar lavage fluid of guinea pigs in each group after administration of Shujinsan was significantly inhibited.

[0138] As Figure 15As shown, compared with the Control group, the mRNA levels of the neurogenic inflammatory factors CGRP and SP in the lung, trachea, and dorsal root ganglion tissues of the guinea pigs in the model group were significantly increased; after administration of different doses of Shujin San, the expression levels of the neurogenic inflammatory factors CGRP and SP in the lung, trachea, and dorsal root ganglion tissues of the guinea pigs were significantly decreased.

[0139] The levels of neurogenic inflammatory factors in the serum and bronchoalveolar lavage fluid of CVA guinea pigs were further detected. As Figure 16 shown by the results, compared with the rats in the Control group, the levels of the neurogenic inflammatory factors CGRP and SP in the serum and bronchoalveolar lavage fluid of the guinea pigs in the model group were significantly increased; compared with the guinea pigs in the model group, the increase in the levels of the neurogenic inflammatory factors CGRP and SP in the serum and bronchoalveolar lavage fluid of each group of guinea pigs after administration of Shujin San was significantly inhibited.

[0140] IV. Result Discussion

[0141] Shujin San can significantly improve the weight loss of guinea pigs after combined modeling with ovalbumin and capsaicin, and reduce the number of coughs; pathological staining found that administration of different doses of Shujin San can significantly reduce the increase in blood vessels, congestion, and infiltration of inflammatory cells under the tracheal epithelium in the lungs, and the thickening of the gland wall was significantly improved; the results of Micro CT showed that the lesion area in the lungs of guinea pigs decreased significantly after administration of different doses of Shujin San; the results of Masson staining showed that the deposition of collagen fibers in the lung and trachea tissue structures of guinea pigs decreased after administration of different doses of Shujin San; AB-PAS staining proved that administration of different doses of Shujin San to the trachea and lung tissues can significantly inhibit the secretion of epithelial mucin; the numbers of white blood cells, neutrophils, eosinophils, and basophils in the peripheral blood and bronchoalveolar lavage fluid were significantly inhibited; the ELISA results confirmed that Shujin San can inhibit the increase in the levels of the neurogenic inflammatory factors CGRP and SP, and the increase in the contents of the inflammatory factors IL-6, TNFα, and IL-4 was also significantly inhibited; the RT-qPCR results showed that administration of Shujin San can significantly reduce the expression levels of the inflammatory factors TNFα and IL-4 mRNA in the lung and trachea tissues, as well as the expression levels of the neurogenic inflammatory factors CGRP and SP mRNA in the lung, trachea, and dorsal root ganglion tissues.

[0142] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A Chinese medicine composition for treating cough variant asthma, characterized in that: Including bupleurum, ephedra, schizonepeta, bitter almond, platycodon, immature bitter orange, red orange peel, reed root, pinellia, belamcanda, bombyx batryticatus, patchouli and mint.

2. The Chinese medicine composition according to claim 1, characterized in that: The mass ratio of Bupleurum, Ephedra and Schizonepeta is: (4-6): (2-4): (1-3).

3. The Chinese medicine composition according to claim 2, characterized in that: The mass ratio of Bupleurum, Ephedra and Schizonepeta is: (5-6): (3-4): (2-3).

4. The Chinese medicine composition according to claim 1, characterized in that: The mass ratio of bitter almond, immature bitter orange and pinellia is: (2-4): (1-3): (1-2).

5. The Chinese medicine composition according to claim 4, characterized in that: The mass ratio of bitter almond, immature bitter orange and pinellia is: (3-4): (2-3):

2.

6. The Chinese medicine composition according to any one of claims 1 to 5, characterized in that: In parts by weight, it includes 4-6 parts of bupleurum, 2-4 parts of honey ephedra, 1-3 parts of schizonepeta, 2-4 parts of bitter almond, 1-3 parts of platycodon, 1-3 parts of immature bitter orange, 1-2 parts of orange peel, 1-3 parts of reed root, 1-2 parts of pinellia, 3-5 parts of belamcanda, 1-2 parts of white bombyx batryticatus, 1 part of patchouli, and 1 part of mint.

7. The Chinese medicine composition according to claim 6, characterized in that: Calculated by weight, it includes 6 parts of bupleurum, 4 parts of honey ephedra, 3 parts of schizonepeta, 4 parts of bitter almond, 3 parts of platycodon, 3 parts of immature bitter orange, 2 parts of orange peel, 3 parts of reed root, 2 parts of pinellia, 5 parts of belamcanda, 2 parts of bombyx batryticatus, 1 part of patchouli, and 1 part of mint.

8. Use of the Chinese medicine composition according to any one of claims 1 to 7 in the preparation of medicines for treating respiratory diseases.

9. The use according to claim 8, characterized in that: The respiratory disease is cough variant asthma.

10. A drug for treating respiratory diseases, characterized in that: A Chinese medicine composition comprising any one of claims 1 to 7.

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