Preparation method and application of traditional Chinese medicine spray for treating asthma

Abstract

The invention discloses a preparation method and application of a traditional Chinese medicine spray for treating asthma, and belongs to the technical field of traditional Chinese medicine preparations. The stability problem of the traditional Chinese medicine spray is systematically solved through the synergistic effect of alcohol precipitation impurity removal, volatile oil clathration, pH regulation and control and the ternary stabilizer. Pharmacodynamic studies show that the spray can remarkably reduce the inflammatory factor level of asthma model mice and prolong the incubation period of asthma inducing of guinea pigs, has the effect better than that of oral granules, takes effect quickly and is suitable for quick relief in the acute attack period of asthma.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, specifically relating to a method for preparing a traditional Chinese medicine spray for treating asthma, and the application of the spray in the preparation of drugs for treating bronchial asthma. Background Technology

[0002] Bronchial asthma is a chronic inflammatory disease of the airways involving multiple cells (such as eosinophils, mast cells, T lymphocytes, neutrophils, and airway epithelial cells) and cellular components. This chronic inflammation leads to airway hyperresponsiveness, typically resulting in widespread and variable reversible airflow limitation, and causing recurrent episodes of wheezing, shortness of breath, chest tightness, or cough. Asthma is mainly staged into acute exacerbations, chronic persistent asthma, and clinical remission, each with different characteristics and treatment goals. Acute exacerbations are characterized by sudden worsening of symptoms, chronic persistent asthma is characterized by long-term recurrent symptoms, and clinical remission is characterized by an asymptomatic or stable state.

[0003] Asthma falls under the category of "wheezing disease" in Traditional Chinese Medicine (TCM). It is caused by latent phlegm in the lungs, which, when triggered by external pathogens, obstructs the airways, impairs the lung's descending function, and leads to the struggle between phlegm and qi, resulting in paroxysmal wheezing and shortness of breath. Clinically, it is characterized by wheezing in the throat, shortness of breath, and even difficulty lying flat. Phlegm in the lungs is both the underlying cause of asthma and a key pathological factor in its onset. Phlegm production is attributed to external pathogens, improper diet, emotional distress, congenital deficiencies, and lung, spleen, and kidney deficiency, which prevent the lungs from distributing body fluids, the spleen from transporting essence, and the kidneys from transforming fluids. This leads to the accumulation of body fluids into phlegm, which then accumulates in the lungs and becomes crucial to the disease. Phlegm is a yin pathogen, easily obstructing the yang of the chest and lungs, hindering the flow of qi, and causing phlegm and qi to congest the airways, leading to asthma attacks. Therefore, chest (lung) yang stagnation and phlegm-qi stagnation are the main pathological features of asthma attacks. Therefore, in response to the pathological characteristics of asthma with insufficient Yang in the chest and phlegm and Qi stagnation, the treatment should focus on relieving chest tightness and regulating Qi, promoting Yang and dispersing stagnation, and resolving phlegm and relieving spasms.

[0004] Currently, Western medicines for treating bronchial asthma mainly include bronchodilators (such as salbutamol) and glucocorticoids. Although they have a rapid onset of action, long-term use may lead to problems such as palpitations, drug tolerance, and systemic side effects. Traditional Chinese medicine decoctions or granules, while having a good overall regulatory effect, have a relatively slow onset of action and limited immediate relief during acute attacks.

[0005] This invention's prescription originates from Professor Wang Peng's experienced formula for treating asthma—the "Anti-Asthma and Relieves Asthma Formula"—developed over many years by a renowned TCM doctor in Hubei Province and the Department of Pulmonary Diseases at Hubei Provincial Hospital of Traditional Chinese Medicine. This formula is a modification of three other formulas: Gualou Xiebai Banxia Decoction, Shaoyao Gancao Decoction, and Xiao Chaihu Decoction. It possesses the effects of regulating qi and relieving chest tightness, promoting yang and dispersing stagnation, and resolving phlegm and relieving spasms. This formula has been used clinically at Hubei Provincial Hospital of Traditional Chinese Medicine for over 20 years, establishing a solid clinical foundation. Clinically, this formula has proven effective and safe in treating acute asthma attacks. Studies have shown that the Anti-Asthma and Relieves Asthma Formula has significant therapeutic effects on asthma model animals. It can promote apoptosis of eosinophils in the lung tissue of asthmatic rats, regulate Fas and Bcl-2 gene expression, reduce serum IgE, IL-5, and IL-13 levels in asthmatic rats, inhibit Th2-type immune responses, reduce airway hyperresponsiveness, and relax airway smooth muscle. Several publications have already explored its effects. [1-3] .

[0006] Traditional Chinese medicine sprays, as a novel dosage form, combine the holistic concept of traditional Chinese medicine with the targeted drug delivery advantages of modern pharmaceutics. Administered directly through inhalation into the lungs, they enable the drug to quickly reach the lesion, exhibiting characteristics such as rapid onset of action, high bioavailability, small dosage, and few side effects. They are particularly suitable for the rapid relief of acute asthma attacks.

[0007] However, the components of traditional Chinese medicine extracts are complex and prone to precipitation. The active ingredients are easily degraded by pH, light, metal ions, and oxygen. Therefore, solving the stability problem of liquid formulations and achieving rapid onset of action are urgent technical challenges in this field.

[0008] References: [1] Pan, Fengman. Effects of Zhixiao Pingchuan Formula on Apoptosis and Regulation of Gene Expression of Eosinophils in Asthmatic Rats [D]. Wuhan: Hubei University of Traditional Chinese Medicine, 2009. [2] Hu Zuowei et al. Experimental study on the effect of traditional Chinese medicine anti-asthmatic and anti-bronchodilator formula on airway inflammation in guinea pig model of asthma. Journal of Guangzhou University of Chinese Medicine. January 2004, Vol. 21, No. 1. [3] Pan Fengman et al. Effects of Zhixiao Pingchuan Formula on EOS in whole blood and bronchoalveolar lavage fluid of asthmatic rats. Sichuan Journal of Traditional Chinese Medicine. Vol. 32, No. 5, 2014. Summary of the Invention The purpose of this invention is to provide a method for preparing a traditional Chinese medicine spray for treating asthma, which improves the efficacy of traditional Chinese medicine through dosage form improvement, while solving the problems of poor formability and stability of traditional Chinese medicine liquid preparations.

[0009] The technical solution of the present invention is as follows: A method for preparing a traditional Chinese medicine spray for treating asthma includes the following steps: (1) Extraction of volatile oil: Take Trichosanthes peel, Allium macrostemon, Bupleurum chinense and Citrus aurantium, add 6 to 10 times the amount of water, and extract by steam distillation for 2 to 6 hours. Collect the volatile oil and keep the residue for later use. (2) Water extraction: Combine the residue from step (1) with Pinellia ternata, Belamcanda chinensis, Ephedra sinica, Paeonia lactiflora, Scutellaria baicalensis and Glycyrrhiza uralensis, add 6 to 10 times the amount of water, heat and extract 1 to 3 times, each time for 1 to 2 hours, combine the extracts, concentrate to a relative density of 1.15 to 1.20, and obtain concentrated solution; (3) Alcohol precipitation: Add ethanol to the concentrate until the alcohol content reaches 50-80%, let stand, centrifuge, separate the supernatant, and recover the ethanol until there is no alcohol smell to obtain the alcohol precipitation concentrate; (4) Inclusion of volatile oil: The volatile oil collected in step (1) is stirred with β-cyclodextrin at 45-60℃ for 1-2 hours, refrigerated overnight, filtered, and dried to obtain the volatile oil inclusion complex. (5) Solution preparation: Add the inclusion complex from step (4) to the alcohol precipitation concentrate from step (3), dilute with water, adjust the pH to 5.0-6.0, add stabilizer, and make up to volume to obtain the drug solution; (6) Fine filtration and filling: The medicine solution is filtered through a microporous membrane and filled into a spray bottle to obtain the product.

[0010] The weight proportions of each Chinese medicinal herb are as follows: Trichosanthes peel 100-150 parts, Allium macrostemon 100-150 parts, Pinellia ternata 100-150 parts, Belamcanda chinensis 120-180 parts, Ephedra sinica 50-80 parts, Paeonia lactiflora 100-150 parts, Bupleurum chinense 100-150 parts, Scutellaria baicalensis 100-150 parts, Citrus aurantium 100-150 parts, Glycyrrhiza uralensis 100-150 parts.

[0011] Preferably, in step (1), the parameters for volatile oil extraction are as follows: the medicinal material is pulverized through a 24-mesh sieve, soaked for 2 hours, 6 times the amount of water is added, and distilled for 4 hours. Experiments show that these parameters are beneficial to improving the extraction efficiency of volatile oil.

[0012] Preferably, in step (2), the parameters for water extraction are: add 8 times the amount of water, extract twice, 1.5 hours each time. Experiments show that these parameters are beneficial to improving the extraction efficiency of active ingredients in traditional Chinese medicine.

[0013] Preferably, in step (3), the alcohol content of the alcohol precipitation is 70%. Experiments show that this parameter condition is conducive to enriching the active ingredients.

[0014] Preferably, in step (4), the β-cyclodextrin is 5-10 times the mass of the volatile oil, more preferably 8 times. Experiments show that these parameters are beneficial for improving the inclusion efficiency of the volatile oil.

[0015] Preferably, in step (4), the parameters for the inclusion of the volatile oil are: inclusion temperature 50°C, and stirring for 1.5 hours. Experiments show that these parameters are beneficial for improving the inclusion rate of the volatile oil.

[0016] Preferably, in step (5), the stabilizer is a combination of sodium bisulfite, ascorbic acid, and EDTA-2Na, with addition amounts of 0.05%, 0.02%, and 0.02%, respectively. Experiments show that this stabilizer combination is beneficial to improving the chemical stability of the active ingredient.

[0017] The present invention also provides a traditional Chinese medicine spray prepared according to the above method, and the application of the spray in the preparation of drugs for treating bronchial asthma.

[0018] The beneficial effects of this invention are: Traditional Chinese medicine (TCM) theory holds that oral medications, through the spleen and stomach's digestive system, allow the medicinal components to be absorbed via the digestive tract, enter the bloodstream, and distribute throughout the body. This allows for the simultaneous regulation of multiple organ functions, including the lungs, spleen, and kidneys, thus exerting a holistic therapeutic effect. Inhalers, considered a Western medicine delivery method, act directly on the airways and lungs, aligning with the theories of "the lungs opening to the nose" and "the lungs governing qi and respiration," allowing the medicine to directly reach the affected area, consistent with the TCM principle of "treating the symptoms in acute cases." Therefore, both dosage forms have their advantages in treating asthma. This invention retains the TCM principles of "regulating qi and relieving chest congestion, promoting yang and dispersing stagnation," while leveraging the advantages of inhalers—"directly reaching the affected area and taking effect quickly"—creatively forming a novel drug delivery mode that combines "local treatment with holistic regulation," achieving unexpected technical results. Pharmacodynamic studies have shown that the inhaler of this invention can significantly reduce the levels of inflammatory factors and eosinophils in asthma model mice and significantly prolong the latency period of asthma induction in guinea pigs, with effects superior to oral granules.

[0019] This invention improves the therapeutic effect of asthma through volatile oil extraction, especially enhancing the anti-inflammatory effect against sensitizing asthma. Furthermore, this invention systematically solves the stability problem of traditional Chinese medicine sprays through a combination of alcohol precipitation, volatile oil encapsulation, pH adjustment, and a ternary stabilizer, effectively ensuring the efficacy of the drug. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments.

[0021] Example 1: Preparation of Compound Anti-Asthma Spray 1. Prescription Medicinal materials: Trichosanthes peel 125g, Allium macrostemon 125g, Pinellia ternata 125g, Belamcanda chinensis 150g, Ephedra sinica 62.5g, Paeonia lactiflora 125g, Bupleurum chinense 125g, Scutellaria baicalensis 125g, Citrus aurantium (fried with wheat bran) 125g, Glycyrrhiza uralensis 125g.

[0022] Additional ingredients: Purified water, 95% ethanol, β-cyclodextrin, Tween-80, steviol glycosides, sodium benzoate, 10% sodium hydroxide solution, sodium bisulfite, ascorbic acid, EDTA-2Na.

[0023] 2. Preparation process (1) Extraction of volatile oil: Take the prescribed amount of four medicinal materials: Trichosanthes peel, Allium macrostemon, Bupleurum chinense, and Citrus aurantium stir-fried with wheat bran. Grind them and pass them through a 24-mesh sieve. Soak them for 2 hours and add 6 times the amount of water (about 3 kg). Extract the volatile oil by steam distillation for 4 hours. Collect 1.55 g of volatile oil with a yield of 0.31%. Keep the collected volatile oil for later use and leave the residue for subsequent water extraction. (2) Water extraction: Combine the dregs from step (1) with the prescribed amounts of Pinellia ternata, Belamcanda chinensis, Ephedra sinica, Paeonia lactiflora, Scutellaria baicalensis, and Glycyrrhiza uralensis, soak in water for 0.5 h, add 8 times the amount of water (about 10 kg), heat and extract twice, 1.5 h each time, combine the extracts, and concentrate under reduced pressure to a relative density of 1.15-1.20 (60 °C) to obtain a concentrated solution; (3) Alcohol precipitation: Slowly add 95% ethanol to the concentrate while stirring, so that the alcohol content reaches 70%, let stand for 24 hours to allow the precipitate to precipitate completely, centrifuge to separate, take the supernatant, and recover the ethanol until there is no alcohol smell to obtain the alcohol precipitation concentrate. (4) Inclusion of volatile oil: Take 12.4g of β-cyclodextrin, add purified water to make a saturated aqueous solution, add 1.55g of volatile oil collected in step (1) at 50℃, keep warm and stir for 1.5h, refrigerate overnight, filter, collect filter cake, dry at 40℃ to obtain about 13.5g of volatile oil inclusion complex, with an inclusion rate of 68.5%; (5) Solution preparation: Add the inclusion complex from step (4) to the alcohol precipitation concentrate from step (3), add purified water to about 800 mL, adjust the pH of the solution to 5.5 with 10% sodium hydroxide solution, add sodium bisulfite 0.5 g, ascorbic acid 0.2 g, and EDTA-2Na 0.2 g in sequence, stir to dissolve, and finally add appropriate amounts of Tween-80, steviol glycosides, and sodium benzoate, and make up to 1000 mL with purified water; (6) Fine filtration and filling: The medicine solution is finely filtered through a 10μm microporous membrane and filled into spray bottles with a volume of 10mL per bottle to obtain compound anti-asthmatic spray.

[0024] Example 2: Preparation of Compound Anti-Asthma Spray 1. Prescription Same as Example 1.

[0025] 2. Preparation process (1) Extraction of volatile oil: Take the prescribed amount of four medicinal materials: Trichosanthes peel, Allium macrostemon, Bupleurum chinense, and Citrus aurantium stir-fried with wheat bran. Grind them and pass them through a 10-mesh sieve. Soak them for 1 hour and add 8 times the amount of water (about 4 kg). Extract the volatile oil by steam distillation for 6 hours. Collect about 1.31 g of volatile oil, with a yield of 0.26%. Keep the collected volatile oil for later use and leave the residue for subsequent water extraction. (2) Water extraction: Combine the dregs from step (1) with the prescribed amounts of Pinellia ternata, Belamcanda chinensis, Ephedra sinica, Paeonia lactiflora, Scutellaria baicalensis, and Glycyrrhiza uralensis, soak in water for 0.5 h, add 6 times the amount of water (about 7.5 kg), heat and extract 3 times, 1 h each time, combine the extracts, concentrate under reduced pressure to a relative density of 1.15-1.20 (60℃) to obtain a concentrated solution; (3) Alcohol precipitation: Slowly add 95% ethanol to the concentrate while stirring until the alcohol content reaches 50%. Let it stand for 24 hours to allow the precipitate to precipitate completely. Centrifuge to separate the precipitate, take the supernatant, and recover the ethanol until there is no alcohol smell to obtain the alcohol precipitation concentrate. (4) Inclusion of volatile oil: Take 13.1g of β-cyclodextrin, add purified water to make a saturated aqueous solution, add 1.31g of volatile oil collected in step (1) at 60℃, keep warm and stir for 1h, refrigerate overnight, filter, collect filter cake, dry at 40℃ to obtain about 13.9g of volatile oil inclusion complex, with an inclusion rate of 59.3%; (5) Solution preparation: Add the inclusion complex from step (4) to the alcohol precipitation concentrate from step (3), add purified water to about 800 mL, adjust the pH of the solution to 5.5 with 10% sodium hydroxide solution, add sodium bisulfite 0.5 g, ascorbic acid 0.2 g, and EDTA-2Na 0.2 g in sequence, stir to dissolve, and finally add appropriate amounts of Tween-80, steviol glycosides, and sodium benzoate, and make up to 1000 mL with purified water; (6) Fine filtration and filling: The medicine solution is finely filtered through a 10μm microporous membrane and filled into spray bottles with a volume of 10mL per bottle to obtain compound anti-asthmatic spray.

[0026] Example 3: Preparation of Compound Anti-Asthma Spray 1. Prescription Same as Example 1.

[0027] 2. Preparation process (1) Extraction of volatile oil: Take the prescribed amount of four medicinal materials: Trichosanthes peel, Allium macrostemon, Bupleurum chinense, and Citrus aurantium stir-fried with wheat bran. Grind them and pass them through a 30-mesh sieve. Soak them for 0.5 hours and add 10 times the amount of water (about 5 kg). Extract the volatile oil by steam distillation for 2 hours. Collect about 1.10 g of volatile oil, with a yield of 0.22%. Keep the collected volatile oil for later use and leave the residue for subsequent water extraction. (2) Water extraction: Combine the dregs from step (1) with the prescribed amounts of Pinellia ternata, Belamcanda chinensis, Ephedra sinica, Paeonia lactiflora, Scutellaria baicalensis, and Glycyrrhiza uralensis, soak in water for 0.5 h, add 10 times the amount of water (about 12 kg), heat and extract once for 2 h, concentrate the extract under reduced pressure to a relative density of 1.15-1.20 (60 °C) to obtain a concentrated solution; (3) Alcohol precipitation: Slowly add 95% ethanol to the concentrate while stirring until the alcohol content reaches 80%. Let it stand for 24 hours to allow the precipitate to precipitate completely. Centrifuge to separate the precipitate, take the supernatant, and recover the ethanol until there is no alcohol smell to obtain the alcohol precipitation concentrate. (4) Inclusion of volatile oil: Take 5.5g of β-cyclodextrin, add purified water to make a saturated aqueous solution, add 1.1g of volatile oil collected in step (1) at 45℃, keep warm and stir for 2h, refrigerate overnight, filter, collect filter cake, dry at 40℃ to obtain about 6.3g of volatile oil inclusion complex, with an inclusion rate of 51.2%; (5) Solution preparation: Add the inclusion complex from step (4) to the alcohol precipitation concentrate from step (3), add purified water to about 800 mL, adjust the pH of the solution to 5.5 with 10% sodium hydroxide solution, add sodium bisulfite 0.5 g, ascorbic acid 0.2 g, and EDTA-2Na 0.2 g in sequence, stir to dissolve, and finally add appropriate amounts of Tween-80, steviol glycosides, and sodium benzoate, and make up to 1000 mL with purified water; (6) Fine filtration and filling: The medicine solution is finely filtered through a 10μm microporous membrane and filled into spray bottles with a volume of 10mL per bottle to obtain compound anti-asthmatic spray.

[0028] Comparative Example 1 Except for the absence of step (3) in the alcohol precipitation process, the formula and other processes and parameters are the same as in Example 1.

[0029] Comparative Example 2 Except for the absence of steps (1) and (4) in the volatile oil extraction and inclusion process, the formulation and other processes and parameters are the same as in Example 1.

[0030] Comparative Example 3 Except for replacing the volatile oil inclusion complex and alcohol precipitation concentrate in steps (5) and (6) of Example 1 with starch and dextrin granulation, the other processes are the same as in Example 1, and compound anti-asthmatic granules are prepared.

[0031] Test case 1. Determination of the content of the spray Method for determining the yield of dry extract: Take 10g of the extract concentrate from Examples 1-3 and Comparative Example 1, weigh it accurately, place it in an evaporating dish dried to constant weight, place it in a water bath to evaporate to dryness, dry it at 100-105℃ to constant weight, transfer it to a desiccator, cool it for 30 minutes, weigh it to obtain the weight of the dry extract, and calculate the total weight of the dry extract in the extract concentrate based on the weight of the dry extract of 10g concentrate.

[0032] Determination of baicalin and ephedrine content: Baicalin and ephedrine in the finished sprays of Examples 1-3 and Comparative Example 1 were determined according to the HPLC method in the Chinese Pharmacopoeia. The results are shown in Table 1 below.

[0033] Table 1: Total dry paste content and index component content in sprays

[0034] The comparison results between Example 1 and Comparative Example 1 show that, under the same prescription of medicinal materials, the total amount of dry extract obtained in Examples 1-3 is significantly lower than that in Comparative Example 1. While the contents of baicalin and ephedrine are slightly lower than in Comparative Example 1, the difference is not significant. This indicates that the alcohol precipitation process effectively reduces impurities in the product while preserving the active ingredients well. The comparison results between Examples 1, 2, and 3 show that Example 1 has higher contents of baicalin and ephedrine. Therefore, the optimal parameters for the water extraction and alcohol precipitation process are: add 8 times the amount of water, extract twice, 1.5 hours each time, and the alcohol content in the precipitated product is 70%.

[0035] 2. Stability study of the spray The prepared spray was placed in a 40℃ stability test chamber for 6 consecutive months. The clarity of the solution was observed, and the onset time of precipitation was recorded. After the observation period, the contents of baicalin and ephedrine in the spray were measured, and the degradation rate was calculated based on the initial contents. The results are shown in Table 2 below.

[0036] Table 2: Stability test results of Examples 1-3 and Comparative Example 1

[0037] The results showed that, compared with Comparative Example 1, Examples 1-3 did not produce significant precipitation during the stability study period, and the contents of baicalin and ephedrine were relatively stable. This indicates that reducing impurities in the liquid system is beneficial to improving the physical and chemical stability of the spray.

[0038] In addition, since the pH and type of stabilizer in the solution preparation process also play an important role in the stability of the spray, we also conducted a screening and comparison, and the results are as follows.

[0039] (1) Effect of different pH values ​​on the stability of sprays Five batches of samples were prepared according to the preparation process described in Example 1. The pH value of the drug solution was adjusted to 3.5-7.5. The onset time of precipitation, the degradation rate of baicalin and ephedrine were used as evaluation indicators. The samples were placed in a stability test chamber at 40℃ for 6 consecutive months to investigate the effect of different pH values ​​on the stability of the drug solution. The results are shown in Table 3 below.

[0040] Table 3: Effect of different pH values ​​on the stability of the spray.

[0041] The results showed that in a liquid environment, as alkalinity increased, the glycosidic bonds of baicalin began to break, the degradation rate accelerated, and the resulting baicalein had low solubility, causing the solution to become turbid. Additionally, ephedrine also showed a tendency for accelerated degradation under neutral and alkaline conditions, which may be related to the oxidation of its benzylic carbon atom. Based on the experimental results, the pH of this product should be controlled under acidic conditions, but excessively low pH can cause mucosal irritation. Therefore, considering both safety and stability, it was ultimately determined that controlling the pH of the spray at 5.0-6.0 is optimal.

[0042] (2) Effect of different stabilizers on the stability of sprays Samples were prepared using different stabilizers according to the formulation and process of Example 1 to investigate the effect of different stabilizer compositions on the stability of the drug solution. The results are shown in Table 4 below.

[0043] Table 4: Effects of different stabilizers on the stability of sprays

[0044] Results analysis: Sodium bisulfite is a strong reducing agent that consumes oxygen; ascorbic acid acts as a free radical scavenger and regenerator, and the combined use of the two has a more significant antioxidant effect. The solution of this invention contains trace amounts of metal ions (such as Fe). 3+ Cu 2+ These are strong oxidizing catalysts, and the addition of the metal ion chelating agent EDTA-2Na can inhibit the catalytic reaction. The three stabilizers work synergistically to improve the chemical stability of the spray.

[0045] 3. Pharmacodynamic evaluation of the spray The therapeutic effect of the inhaler on asthma was evaluated using a mouse asthma model and animal bronchodilator experiments.

[0046] (1) Effect on ovalbumin (OVA)-induced mouse asthma model For modeling with OVA: Weigh 2 mg of OVA and dissolve it in PBS solution containing 20 mg / mL aluminum hydroxide adjuvant, to a final concentration of 100 μg / mL. For 1% OVA saline solution: Weigh 1 g of OVA and dissolve it in 100 mL of saline solution, to a final concentration of 1%.

[0047] Sensitization phase: D0, D7, and D14. Except for the blank control group, which received intraperitoneal injection of normal saline, all other groups received intraperitoneal injection of OVA aluminum hydroxide adjuvant at a dose of 100 μg / kg. Challenge phase: D21-D28. Except for the blank control group, all other groups received 1% OVA normal saline solution via ultrasonic nebulization. The grouping and administration regimens are shown in Table 5 below.

[0048] Table 5: Animal grouping and administration

[0049] Twenty-four hours after the final stimulation, blood was collected from the eyeballs to measure serum immunoglobulin E (IgE) levels; bronchoalveolar perfusion fluid was collected to measure TNF-α levels and observe the number of eosinophils. The results are shown in Table 6 below.

[0050] Table 6: Effects of the inhaler on cytokines in bronchoalveolar lavage fluid of OVA-induced asthma model mice

[0051] Note: n=10, mean±SD, compared with the blank control group. p <0.05; compared with the model group, # p <0.05; compared with Example 1 group, △ p <0.05.

[0052] The results showed that Example 1 and Comparative Examples 2 and 3 significantly improved the pathological state of the OVA-induced asthma model and reduced the levels of inflammatory factors and effector cells. Specifically, the levels of TNF-α and eosinophils in Example 1 were significantly lower than those in Comparative Example 2, indicating that the volatile components in the formula could, to some extent, promote the anti-inflammatory effect of the traditional Chinese medicine. Furthermore, no statistically significant differences were found in the various inflammatory indicators between Example 1 and Comparative Example 3 (possibly because the local advantage of the inhaler is not significant in chronic inflammation), but the numerical values ​​still showed that the anti-inflammatory effect of Example 1 was superior to that of Comparative Example 3.

[0053] (2) Study on the antiasthmatic effect A mixture of 2% acetylcholine chloride and 0.4% histamine phosphate was prepared using physiological saline as an induction solution. Guinea pigs were placed in a YLS-8A multifunctional cough and asthma induction device, and the induction solution was sprayed into the hood for 15 seconds using an ultrasonic nebulizer. The latency period of induction was observed, i.e., the time from the start of the spray to the onset of asthma until convulsions and falls. Guinea pigs with a latency period <150 seconds were grouped. After 3 days of administration, one hour after the last administration, the guinea pigs were placed in the YLS-8A multifunctional cough and asthma induction device, and the nebulized mixture of 2% acetylcholine chloride and 0.4% histamine phosphate was sprayed into the hood. The latency period of induction was then measured. The grouping and administration regimens are shown in Table 7 below.

[0054] Table 7: Animal grouping and administration

[0055] The test results are shown in Table 8.

[0056] Table 8: Effects of spray on the latency period of asthma in guinea pigs

[0057] Note: n=10, mean±SD, compared with the blank control group. p<0.05; compared with the model group, #p<0.05, compared with Example 1 group, △ p <0.05.

[0058] The results showed that Example 1 and Comparative Examples 2 and 3 significantly prolonged the latency of asthma induced by acetylcholine chloride and histamine phosphate. Example 1 showed a significant difference compared with Comparative Examples 2 and 3. This demonstrates that volatile oil is the key active ingredient in the antiasthmatic and bronchodilator formula for treating asthma, and that the inhaler has the advantages of local targeting and rapid onset of action, making it more beneficial for asthma treatment than oral formulations.

Claims

1. A method for preparing a traditional Chinese medicine spray for treating asthma, characterized in that... Includes the following steps: (1) Extraction of volatile oil: Take Trichosanthes peel, Allium macrostemon, Bupleurum chinense and Citrus aurantium, add 6 to 10 times the amount of water, and extract by steam distillation for 2 to 6 hours. Collect the volatile oil and keep the residue for later use. (2) Water extraction: Combine the residue from step (1) with Pinellia ternata, Belamcanda chinensis, Ephedra sinica, Paeonia lactiflora, Scutellaria baicalensis and Glycyrrhiza uralensis, add 6 to 10 times the amount of water, heat and extract 1 to 3 times, each time for 1 to 2 hours, combine the extracts, concentrate to a relative density of 1.15 to 1.20, and obtain concentrated solution; (3) Alcohol precipitation: Add ethanol to the concentrate until the alcohol content reaches 50-80%, let stand, centrifuge, separate the supernatant, and recover the ethanol until there is no alcohol smell to obtain the alcohol precipitation concentrate; (4) Inclusion of volatile oil: The volatile oil collected in step (1) is stirred with β-cyclodextrin at 45-60℃ for 1-2 hours, refrigerated overnight, filtered, and dried to obtain the volatile oil inclusion complex. (5) Solution preparation: Add the inclusion complex from step (4) to the alcohol precipitation concentrate from step (3), dilute with water, adjust the pH to 5.0-6.0, add stabilizer, and make up to volume to obtain the drug solution; (6) Fine filtration and filling: The medicine solution is filtered through a microporous membrane and filled into a spray bottle to obtain the product.

2. The preparation method according to claim 1, characterized in that, The weight proportions of each Chinese medicinal herb are as follows: Trichosanthes peel 100-150 parts, Allium macrostemon 100-150 parts, Pinellia ternata 100-150 parts, Belamcanda chinensis 120-180 parts, Ephedra sinica 50-80 parts, Paeonia lactiflora 100-150 parts, Bupleurum chinense 100-150 parts, Scutellaria baicalensis 100-150 parts, Citrus aurantium 100-150 parts, Glycyrrhiza uralensis 100-150 parts.

3. The preparation method according to claim 1, characterized in that, In step (1), the parameters for extracting the volatile oil are as follows: the medicinal material is pulverized through a 24-mesh sieve, soaked for 2 hours, 6 times the amount of water is added, and distilled for 4 hours.

4. The preparation method according to claim 1, characterized in that, In step (2), the parameters for water extraction are: add 8 times the amount of water, extract twice, and each time for 1.5 hours.

5. The preparation method according to claim 1, characterized in that, In step (3), the alcohol content of the alcohol precipitation is 70%.

6. The preparation method according to claim 1, characterized in that, In step (4), the β-cyclodextrin is 5-10 times the mass of the volatile oil, preferably 8 times.

7. The preparation method according to claim 6, characterized in that, In step (4), the parameters for the inclusion of volatile oil are: inclusion temperature 50°C, and stirring for 1.5 hours.

8. The preparation method according to claim 1, characterized in that, In step (5), the stabilizer is a combination of sodium bisulfite, ascorbic acid and EDTA-2Na, with addition amounts of 0.05%, 0.02% and 0.02%, respectively.

9. A traditional Chinese medicine spray for treating asthma, prepared according to any one of claims 1-8.

10. The use of the traditional Chinese medicine spray according to claim 9 in the preparation of a drug for treating bronchial asthma.

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