Traditional Chinese medicine extract for treating bronchial asthma as well as preparation method and application thereof

Abstract

The invention provides a traditional Chinese medicine extract for treating bronchial asthma and a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine extraction. The preparation method comprises the following steps: soaking rhizoma belamcandae, schisandra chinensis, ginger, jujube and ephedra with water, decocting with water, extracting, soaking ephedra independently, adding rhizoma belamcandae, schisandra chinensis, ginger and jujube, decocting together, and filtering to obtain a total water extract; soaking aster tataricus, tussilago farfara and rhizoma pinellinae praeparata with an ethanol aqueous solution, and performing percolation extraction to obtain a percolate; mixing the total water extract and the percolation liquid, and concentrating to obtain an extract; and soaking the herba asari with water, performing distillation extraction to obtain volatile oil, and mixing the extract and the volatile oil to obtain the rhizoma belamcandae and herba ephedrae decoction extract. The blackberry lily and ephedra decoction extract provided by the invention can remarkably improve release of inflammatory factors and airway remodeling factors related to allergic asthma in an airway, delay airway remodeling, regulate immune balance and inhibit pathological conditions such as overexpression of mucoprotein and inflammatory protein.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a traditional Chinese medicine extract for treating bronchial asthma, and a preparation method and application thereof. Background Art

[0002] Bronchial asthma (hereinafter referred to as asthma) is a common and difficult-to-treat disease worldwide. Asthma is a chronic inflammatory disease of the airways involving multiple cells, which is associated with airway hyperresponsiveness, airway wall remodeling, etc. It is characterized by intermittent attacks, usually at night, manifested by dyspnea, wheezing and coughing. Key indicators for assessing the severity of asthma include increased lung inflammation, goblet cell hyperplasia, peribronchial collagen accumulation, and airway wall thickening. Clinically, asthma patients have elevated levels of lung inflammatory markers and severe infiltration of immune cells even in the asymptomatic period. Studies have shown that the dysregulated interaction between airway mucosal epithelium and innate immune cells has been identified as the main cause of asthma and allergic inflammation.

[0003] At present, the main drugs for the treatment of asthma are corticosteroids, but in clinical applications, a considerable number of patients show resistance to these drugs, resulting in poor disease control and frequent exacerbations. At the same time, patients who take medication for a long time are prone to systemic side effects such as osteoporosis, infection and growth restriction, and have a high recurrence rate after discontinuation of medication. The current concept of asthma treatment has shifted from short-term symptom control to long-term symptom prevention, with the goal of achieving disease remission. Therefore, it is necessary to develop more effective drugs based on traditional Chinese medicine with fewer side effects as a potential supplement to traditional asthma therapy.

[0004] Traditional Chinese medicine has a long history of treating asthma. Shegan Mahuang Decoction was first recorded in the Golden Chamber by Zhang Zhongjing of the Eastern Han Dynasty. It is a classic prescription for treating cold asthma. "Coughing and shortness of breath, water chicken sound in the throat, Shegan Mahuang Decoction is the main treatment." This prescription has been used by many doctors to this day, and the famous doctor Cheng Menxue even praised it as the ancestral prescription for treating asthma. The whole prescription of Shegan Mahuang Decoction contains nine medicinal materials: Shegan, Ephedra, Asarum, Aster, Coltsfoot, Ginger, Schisandra, Jujube, and Pinellia. All the medicines play the role of clearing the lungs and dispersing cold, resolving phlegm and relieving asthma. It has been used clinically for 1,800 years. It has a definite effect on the treatment of asthma, and there has been a certain mechanism of action research, which is worthy of in-depth development and utilization. In order to better develop the effective component group of the compound, the present invention explores the extraction process of Shegan Mahuang Decoction, in order to prepare a Shegan Mahuang Decoction extract with better efficacy. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a Shegan Mahuang decoction extract, so as to effectively extract the effective therapeutic ingredients in the Shegan Mahuang decoction and improve the therapeutic effect on bronchial asthma.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a Shegan Mahuang decoction extract, comprising the following steps:

[0008] 1) Take Belamcanda chinensis, Schisandra chinensis, ginger and jujube, and soak them in water;

[0009] 2) Soaking ephedra in water and boiling separately, adding the soaked Chinese medicine and water in step 1), boiling together, separating the solid and the liquid, and obtaining the first extract;

[0010] 3) taking the residue after solid-liquid separation in step 2) and boiling it with water, separating the solid and liquid, collecting the extract and combining it with the first extract to obtain a total water extract;

[0011] 4) taking Aster tataricus, Coltsfoot flos, and Pinellia ternata, soaking them in an ethanol solution, performing percolation, and collecting the percolation liquid;

[0012] 5) mixing the total water extract and the percolate, and concentrating them to obtain an extract;

[0013] 6) Soak the asarum in water and then distill it to extract the volatile oil;

[0014] 7) mixing the extract with the volatile oil to obtain the Shegan Mahuang decoction extract.

[0015] Preferably, the weight proportions of the raw materials for preparing the Shegan Mahuang Decoction extract are: 7-10 parts of the Belamcanda, 10-14 parts of Ephedra, 10-14 parts of Schisandrae Chinensis, 10-14 parts of Ginger, 20-30 parts of Jujube, 7-10 parts of Aster, 7-10 parts of Coltsfoot Flos, 10-14 parts of Pinellia and 7-10 parts of Asarum.

[0016] Preferably, the amount of water used for soaking in step 1) is 8 to 10 times the total mass of Belamcanda chinensis, Schisandra chinensis, ginger and jujube, and the soaking time is 0.5 to 1 hour.

[0017] Preferably, the amount of water used for soaking in step 2) is 15 to 20 times the mass of ephedra, the soaking time is 0.5 to 1 hour, the time for boiling separately is 10 to 20 minutes, and the time for boiling together is 0.5 to 1 hour.

[0018] Preferably, the amount of water added in step 3) is equal to the sum of the amount of water used for soaking in steps 1) and 2), the decoction is performed 1 to 2 times, each time for 0.5 to 1 hour, and the extract and the residue are separated after each decoction, the residue is used for decoction again, and the extract is collected for later use.

[0019] Preferably, the volume concentration of the ethanol solution in step 4) is 60-65%, the amount of ethanol used during the soaking is 3-5 times the total mass of Aster tataricus, Coltsfoot flos and Pinellia ternata, the filtration uses an ethanol solution with a volume concentration of 60-65% as an extractant, the amount used is 8-12 times the total mass of Aster tataricus, Coltsfoot flos and Pinellia ternata, the flow rate of the filtration is 0.7-1.2 mL / min, and the temperature is 15-25°C.

[0020] Preferably, the amount of water used for soaking in step 6) is 15 to 20 times the mass of Asarum, the soaking time is 0.5 to 1 hour, and the distillation extraction time is 4 to 6 hours.

[0021] The present invention also provides a Belamcanda chinensis decoction extract prepared according to the above preparation method.

[0022] The present invention also provides a use of the Shegan Mahuang decoction extract in preparing a medicine for treating bronchial asthma.

[0023] Preferably, the drug is a liquid preparation or a solid preparation containing pharmaceutically acceptable excipients; the liquid preparation is a decoction, a suspension or an emulsion, and the solid preparation is a tablet, a capsule, a powder, a pill or a granule.

[0024] Compared with other extraction methods, the extract of Shegan Mahuang Tang prepared by the method of the present invention can significantly improve the release of allergic asthma-related inflammatory factors and airway remodeling factors in the airway, delay airway remodeling, and improve the pathological conditions of collagen fiber deposition, goblet cell metaplasia, regulating immune balance, and inhibiting the overexpression of mucin and inflammatory proteins in asthmatic mice. The Shegan Mahuang Tang extract of the present invention has stable quality, safe use, easy control, and a simple preparation method, which is convenient for production and clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A is a HE staining image of the lung tissue (small airways, diameter less than 300 μm) of each group of mice in Example 6 (magnification: 400×); B is a statistical graph of the airway wall thickness of the lung tissue (small airways, diameter less than 300 μm) of each group of mice in Example 6;

[0026] Figure 2 A is a HE staining image of the lung tissue (large airways, diameter greater than 300 μm) of each group of mice in Example 6 (magnification: 400×); B is a statistical graph of the airway wall thickness of the lung tissue (large airways, diameter greater than 300 μm) of each group of mice in Example 6;

[0027] Figure 3A is a PAS staining image of the lung tissue of each group of mice in Example 6 (magnification: 400×); B is a statistical graph of PAS bronchial positivity of the lung tissue of each group of mice in Example 6;

[0028] Figure 4 A is a Masson staining image of the lung tissue of each group of mice in Example 6 (magnification: 400×); B is a statistical result image of the collagen fiber area of ​​the lung tissue of each group of mice in Example 6;

[0029] Figure 5 This is a grayscale analysis diagram of the relevant protein bands in the lung tissues of each group of mice in Example 6;

[0030] Figure 6 1 is a one-way analysis of variance diagram of related proteins in the lung tissues of each group of mice in Example 6, wherein A is a one-way analysis of variance diagram of NF-κB protein; B is a one-way analysis of variance diagram of mTOR protein; C is a one-way analysis of variance diagram of p-NF-κB protein; D is a one-way analysis of variance diagram of MUC5B protein; and E is a one-way analysis of variance diagram of p-mTOR protein;

[0031] Figure 7 The immunofluorescence images of inflammatory proteins in the lung tissues of each group of mice in Example 6;

[0032] Figure 8 Graphs showing the fluorescence intensity of inflammatory proteins in the lung tissues of mice in each group in Example 6, wherein A is a graph showing the fluorescence intensity of NF-κB protein; B is a graph showing the fluorescence intensity of p-NF-κB protein;

[0033] Fig. 9 The results of LTB4 content in lung tissue of each group of mice in Example 6;

[0034] Fig.10 The results of TGF-β content in lung tissue of each group of mice in Example 6;

[0035] Fig.11 The results of IL-4 content in lung tissue of each group of mice in Example 6;

[0036] Fig.12 The results of IFN-γ content in lung tissue of each group of mice in Example 6;

[0037] Fig.13 is the change in the ratio of IL-4 / IFN-γ axis in lung tissue of each group of mice in Example 6;

[0038] Fig.14 The results of IL-10 content in lung tissue of each group of mice in Example 6;

[0039] Fig.15The results of IL-17A content in lung tissue of each group of mice in Example 6;

[0040] Fig.16 The figure shows the changes in the ratio of IL-10 / IL-17A axis in the lung tissue of each group of mice in Example 6. DETAILED DESCRIPTION

[0041] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0042] The various Chinese medicinal materials used in the present invention were purchased from Yunnan Xinglintang Biotechnology Co., Ltd.

[0043] Example 1

[0044] Take 9 grams of dried Belamcanda, 12 grams of Ephedra, 9 grams of Aster, 9 grams of Coltsfoot, 12 grams of Pinellia, 12 grams of Schisandra, 12 grams of Ginger and 28 grams of jujube (7 pieces) and make them into rough blocks. Ephedra needs to be decocted first, so it is soaked in 200 mL of water alone, and the other seven Chinese medicines are soaked in 1200 mL of water together, and the soaking time is 0.5 hours. Take Ephedra and its soaking water and boil them separately, then boil them on low heat for 10 minutes, add the other seven Chinese medicines and water and boil them for 0.5 hours, filter the supernatant to get the first water extract, add 1400 mL of water to decoct the residue, filter to get the extract, repeat 2 times, 0.5 hours each time, and combine the two extracts with the first extract to get the total water extract. The total water extract is concentrated under reduced pressure at 50°C for 4 hours using a rotary evaporator to obtain an extract that can only flow slowly under gravity. Take 9 grams of Asarum and use 135 mL of water as an extractant, soak in a steam distillation device for 0.5 hours, then adjust the volatile oil tester according to the method of the "Chinese Pharmacopoeia" (Pharmacopoeia General Principles 2204), heat with a low heat to keep it slightly boiling for 5 hours, and collect the volatile oil. The extract and the volatile oil are evenly mixed to obtain the extract SM1.

[0045] Example 2

[0046] Take 9g of clean and dried Belamcanda, 12g of Ephedra, 9g of Asarum, 12g of Schisandra, 12g of Ginger and 28g (7 pieces) of jujube and make them into rough blocks. Soak Ephedra alone in 200mL of water, and soak the other five Chinese medicines in 800mL of water for 0.5h. Boil Ephedra and its soaking water separately, boil it on low heat for 10min, add the other seven Chinese medicines and water and boil for 0.5h, filter the supernatant to get the first water decoction extract, add 1000mL of water to decoct the extract filtered from the residue, repeat 2 times, 0.5h each time, combine the two extracts with the first extract to get the total water extract. Take 9 grams of Aster tataricus, 9 grams of Coltsfoot Flos, and 12 grams of Pinellia ternata, which are made into coarse blocks, use a 60% ethanol aqueous solution as an extraction solvent, soak with 100 mL of the solvent for 24 hours, add 300 mL of the solvent and use a percolation device to percolate at a flow rate of 0.83 mL / min to obtain an ethanol percolate. Combine the total water extract and the ethanol percolate, use a rotary evaporator to reduce pressure at 50° C. for 4 hours to recover the solvent, and obtain an extract that can only flow slowly under gravity, that is, extract SM2.

[0047] Example 3

[0048] Take 9g of clean and dried Belamcanda chinensis, 9g of Schisandra chinensis, 12g of ginger and 28g of jujube (7 pieces), cut into coarse pieces, add 600mL of water and soak for 0.5h; take 12g of clean and dried Ephedra sinica, cut into coarse pieces, add 200mL of water and soak for 0.5h, boil separately for 10min, add the soaked Belamcanda chinensis, Schisandra chinensis, ginger and jujube and soaking water, boil together for 0.5h, filter and obtain the first extract; take the filtered residue and add 800mL of water to decoct, filter and obtain the extract, repeat 2 times, each time for 0.5h, combine the two extracts with the first extract to obtain the total water extract; take 9g of Aster tataricus, 9g of Coltsfoot flos and 12g of French Pinellia tuber, add 100mL of 60% ethanol aqueous solution as extractant, soak for 24h, then add 300mL of 60% ethanol solvent for percolation, the flow rate is 0.83mL / min, and obtain ethanol percolation liquid; the total water extract and ethanol percolation liquid are combined, and a rotary evaporator is used to reduce pressure for 4h at 50°C to recover the solvent, and an extract that can only flow slowly under gravity is obtained, 9g of Asarum is taken, 135mL of water is added, and after soaking for 0.5h, a steam distillation device is used for distillation, and a volatile oil tester is adjusted according to the method of "Chinese Pharmacopoeia" (Pharmacopoeia General Principles 2204), and a small fire is heated to keep a slight boil for 5h, and volatile oil is collected. The extract and the volatile oil are evenly mixed to obtain a Belamcanda Ephedra Decoction extract, that is, extract SM3 is obtained.

[0049] Example 4

[0050] Take 9g of clean and dried Belamcanda, 12g of Ephedra, 9g of Aster, 9g of Coltsfoot, 12g of Pinellia, 12g of Schisandra, and 28g (7 pieces) of jujube, and make them into rough blocks. Soak Ephedra alone in 200mL of water, and soak the other six Chinese medicines in 1200mL of water for 0.5 hours. Boil Ephedra and its soaking water separately, and boil it for 10 minutes on low heat. Add the other seven Chinese medicines and water and boil for 0.5 hours. Filter the supernatant to obtain the first decoction extract, add 1400mL of water to decoct the residue and filter to obtain the extract, repeat 2 times, each time for 0.5 hours, and combine the two extracts with the first extract to obtain the total water extract. Take 9 grams of coarse pieces of asarum and 12 grams of ginger, use 60% ethanol aqueous solution as the extraction solvent, soak with 70 mL of solvent for 24 hours, add 200 mL of solvent and use a percolation device for percolation at a flow rate of 0.83 mL / min to obtain ethanol percolation liquid, combine the total water extract and the ethanol percolation liquid, use a rotary evaporator to reduce pressure at 50°C for 4 hours to recover the solvent, and obtain an extract that can only flow slowly under gravity, that is, extract SM4.

[0051] Example 5

[0052] Take 9g of clean and dried Belamcanda, 12g of Ephedra, 9g of Asarum, 9g of Aster, 9g of Coltsfoot, 12g of Pinellia, 12g of Schisandra, 12g of Ginger and 28g (7 pieces) of jujube, and make them into rough pieces. Ephedra needs to be decocted first, so it is soaked in 200mL of water alone, and the other eight Chinese medicines are soaked in 1400mL of water together, and the soaking time is 0.5h. Take Ephedra and its soaking water and boil them separately, then boil them on low heat for 10min, add the other eight Chinese medicines and water and boil them for 0.5h, filter the supernatant to get the first water extract, add 1500mL of water to decoct the residue, filter to get the extract, repeat 2 times, 0.5h each time, and combine the two extracts with the first extract to get the total water extract. The total water extract was concentrated under reduced pressure at 50°C for 4 hours using a rotary evaporator to obtain an extract that could only flow slowly under the action of gravity, namely the Shegan Mahuang decoction water extract SMD.

[0053] Example 6 Therapeutic effect of Shegan Mahuang decoction extract on asthma model

[0054] Experimental animals: 80 healthy SPF-grade Balb / c female mice, aged 6-8 weeks, weighing 18-20 g, were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences and housed in the SPF-grade experimental animal room of Kunming University of Science and Technology.

[0055] The mice were divided into 8 groups, 10 mice in each group. In the following, SMD was the extract of Belamcanda Decoction prepared in Example 5, SM1 was the extract of Belamcanda Decoction prepared in Example 1, SM2 was the extract of Belamcanda Decoction prepared in Example 2, SM3 was the extract of Belamcanda Decoction prepared in Example 3, and SM4 was the extract of Belamcanda Decoction prepared in Example 4;

[0056] The mouse asthma model was established by ovalbumin (OVA) combined with aluminum hydroxide stimulation. Mice were sensitized by intraperitoneal injection of 1% ovalbumin (OVA) solution (w / v) on days 0, 7, and 14, which dissolved 2 mg OVA and 20 mg aluminum hydroxide in 0.2 ml saline. Starting from day 21, mice were stimulated with 2% OVA solution (w / v) by ultrasonic nebulization for 30 minutes every day for 2 weeks. Mice in the CON (Control) group were given an equal amount of saline during sensitization and challenge. From the 21st day to the 35th day, each group of mice was gavaged with 200 microliters 30 minutes before each atomization, the Shegan Mahuang Tang decoction water extract SMD group was given SMD (196 mg / 200 μL / d), the control sample SM1 group was given SM1 (230.4 mg / 200 μL / d), the control sample SM2 group was given SM2 (221 mg / 200 μL / d), the Shegan Mahuang Tang extract SM3 group of the present invention was given SM3 (221.4 mg / 200 μL / d) and the control sample SM4 group was given SM4 (213 mg / 200 μL / d). The control group and the asthma group were gavaged with an equal amount of normal saline, and the positive control group was given dexamethasone intraperitoneally.

[0057] 1. Animal behavioral observation: The overall condition of the mice was tested, and the mental state, weight changes, and behavioral performance of the mice were continuously observed during the experiment.

[0058] It was observed that after two weeks of continuous gavage of the extract of Shegan Mahuang Tang SM3, the mice had a stable body weight, were active and healthy, indicating that the Shegan Mahuang Tang extract was safe and non-toxic. During the administration period, the mice in the SM1 and SM4 treatment groups lost weight significantly after one week of aerosol stimulation treatment, among which the SM4 treatment group lost weight the fastest. Combined with behavioral observations, we noticed that these two groups of mice showed lethargy and limb weakness, indicating that these two groups of extracts may have concentrated more toxic components and are not as safe as SM3. During the experiment, after the aerosol allergens were aerosolized, the mice in the asthma group showed reactions such as continuous scratching of the ears and cheeks, restlessness, arched back and ruffled hair, rapid breathing, and lying motionless; the mice in the control group did not have obvious abnormal reactions; the above reactions of the mice in the dexamethasone treatment and SM2 and SM3 drug intervention groups were significantly lighter.

[0059] 2. Pathological section staining experiment to detect pathological changes in lung tissue

[0060] The mice were killed, and the lung tissues were quickly removed and fixed in tissue fixative for 2 weeks. The sections were embedded in OCT freezing embedding medium and 5-micron sections were made with a Leica slicer. The sections were stained with various staining kits to obtain hematoxylin-eosin (HE) staining, periodic acid-Schiff base (PAS) staining, and Masson trichrome staining. The tissue pathological state was observed under a microscope and images were collected. The positive area was calculated using Fiji ImageJ. The results are as follows:

[0061] HE staining ( Figure 1 and Figure 2 ) The results showed that compared with the blank control group, the asthma group mice had inflammatory cell infiltration around the bronchi of the lung tissue, thickening of the tracheal wall, thickening of the alveolar wall, damaged and detached airway epithelial cells, and rough and uneven airway surface. After administering SM3, an extract from Shegan Mahuang Decoction, the lung pathology of the mice was significantly improved, no inflammatory cell infiltration was observed, the tracheal wall was similar to the blank control, and the airway surface was smooth and flat. The airway wall thickness of small airways (less than 300μm in diameter) and large airways (more than 300μm in diameter) in each group was calculated, and it was found that the average airway wall thickness of the SM3 treatment group was the smallest among the four drug-treated groups, indicating that its effect in inhibiting airway thickening and remodeling is better than SM1, SM2, and SM4.

[0062] PAS staining ( Figure 3 ) The results showed that compared with the blank control group, goblet cell metaplasia appeared in the bronchial lung tissue of mice in the asthma group, and the airway diameter was reduced, which hindered ventilation. The PAS-positive area of ​​each group was calculated and it was found that the goblet cell metaplasia of the lung tissue of mice in the SM3 treatment group was significantly reduced, and the PAS-positive area was the smallest among the four treatment groups, indicating that its effect of inhibiting airway goblet cell metaplasia and airway remodeling was better than SM1, SM2, and SM4.

[0063] Masson staining ( Figure 4 ) The results showed that compared with the blank control group, the asthma group mice had obvious collagen deposition and thickening of fibrous tissue around the bronchus in the lung tissue. The MASSON-positive area of ​​each group was calculated and it was found that the collagen deposition around the bronchus in the lung tissue of the mice treated with SM3 was significantly improved, and the MASSON-positive area was the smallest among the four drug-treated groups, indicating that its effect of inhibiting airway goblet cell metaplasia and airway remodeling was better than SM1, SM2, and SM4.

[0064] 3. Western Blot to detect related protein levels in lung tissue

[0065] The mice were killed, and the lung tissues were quickly removed, frozen in liquid nitrogen, and then stored at -80°C. The frozen mouse lung tissues were taken out, tissue lysis buffer and protease phosphatase inhibitors were added, ground into a homogenate, and centrifuged to extract the total protein of the tissue. After BCA quantitatively determined the total protein concentration, each protein sample was prepared in proportion, and then heated and denatured to obtain the sample protein. 7.5% and 10% SDS-PAGE separation gel and concentration gel were prepared, and an equal amount of protein sample (20μg) was loaded into each well, electrophoresed at 150V until separation, transferred to the membrane at a constant voltage of 110V, and blocked in 5% skim milk at room temperature for 2 hours. After blocking, the primary antibody was incubated at 4°C overnight, washed with TBST the next day, and incubated with the secondary antibody at room temperature for 2 hours. The bands were imaged using the ECL luminescence imaging system, the grayscale value of the protein bands was analyzed by Image J software, and the Graphpad Prism software was used to draw the graph and perform a one-way analysis of variance (ANOVA). p<0.05 indicated a significant difference. The results are as follows:

[0066] Figure 5 and Figure 6 The results showed that compared with the blank control group, the levels of NF-κB, p-NF-κB, mTOR, p-mTOR and MUC5B proteins in the lung tissues of mice in the asthma group were increased; compared with the asthma group, the extract of Shegan Mahuang decoction SM3 could reduce the levels of NF-κB, p-NF-κB, mTOR, p-mTOR and MUC5B proteins in the lungs of asthma mice. For the mucus-related protein MUC5B, the effect of SM3 was stronger than that of all other drug-administered groups. For the inflammatory protein mTOR, the SM3 group was similar to the SMD group in the effect of inhibiting total protein, and the efficacy of the SM3 group in inhibiting its phosphorylated protein was significantly stronger than SM1, SM2, and SM4. For the inflammatory protein NF-κB, the SM3 group was inferior to the SMD group in the results of the WB experiment, but based on the intuitive immunofluorescence staining results of lung tissue, the SM3 group could significantly reduce the expression of NF-κB and its phosphorylated proteins in the lungs, especially in the airways, and was similar to the SMD group.

[0067] 4. Immunofluorescence assay to detect the levels of related inflammatory proteins in lung tissue

[0068] The mice were killed, and the lung tissue was quickly removed and fixed in tissue fixative for 2 weeks. The slices were embedded in OCT freezing embedding medium and made into 20 micron slices with a Leica slicer. The slices were blocked with 3% bovine serum albumin at room temperature for 1 hour. Subsequently, the primary antibody was incubated overnight at 4°C in the dark. After washing with TBST the next day, the fluorescent secondary antibody was incubated at room temperature in the dark for 1 hour. The cell nuclei were counterstained with DAPI and then sealed. Imaging was performed using a NIKON TI-E-AIR automatic inverted fluorescence microscope. The results are as follows:

[0069] Immunofluorescence of lung samples Figure 8 ) The results showed that compared with the blank control group, the levels of NF-κB and p-NF-κB proteins in the lung tissue of the asthma group mice were significantly increased, and were concentrated around the airways; compared with the asthma group, SM3, an extract from Shegan Mahuang decoction, could reduce the levels of NF-κB and p-NF-κB proteins around the airways of the lungs of asthma mice, especially in the inhibition of phosphorylated NF-κB protein, SM3 showed better efficacy than SM1, SM2, and SM4.

[0070] 5. Changes of cytokines in mouse lung tissue

[0071] The mice were killed, and the lung tissues were quickly removed, frozen in liquid nitrogen, and then stored at -80°C. The frozen mouse lung tissues were removed, PBS and protease phosphatase inhibitors were added, ground into a homogenate, and centrifuged to extract the tissue supernatant. The ELISA kit instructions were followed to determine the changes in the levels of LTB4, TGF-β, IL-4, IFN-γ, IL-10, and IL-17A. Graphpad Prism software was used to plot and perform one-way analysis of variance (ANOVA). p<0.05 indicated a significant difference. The results are as follows:

[0072] ELISA of lung samples ( Figures 9 to 16 ) The results showed that compared with the blank control group, the concentrations of leukotriene inflammatory factor LTB4, key factor of airway remodeling TGF-β, Th2 response representative factor IL-4, and Th17 response representative factor IL-17A in the lung tissue of mice in the asthma group were significantly increased; the concentrations of Th1 response representative factor IFN-γ and Treg response representative factor IL-10 were significantly reduced. Compared with the asthma group, the extract SM3 of Shegan Mahuang decoction can significantly reduce the concentrations of LTB4, TGF-β, IL4, and Th17A in the lungs of asthmatic mice, increase the concentrations of IFN-γ and IL-10, regulate the Th1 / Th2 immune balance represented by the IL-4 / IFN-γ axis and the Treg / Th17 immune balance represented by the IL-10 / IL-17A axis, and the effect is better than that of SM1, SM2, and SM4 groups.

[0073] It can be seen from the above examples that the extract SM3 of Shegan Mahuang Decoction provided by the present invention can significantly improve pathological conditions such as pulmonary inflammatory infiltration, airway remodeling, goblet cell metaplasia, and collagen fiber deposition in OVA asthmatic mice.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a Shegan Mahuang decoction extract, characterized in that: The following steps are involved: 1) Take Belamcanda chinensis, Schisandra chinensis, ginger and jujube, and soak them in water; 2) Soaking ephedra in water and boiling separately, adding the soaked Chinese medicine and water in step 1), boiling together, separating the solid and the liquid, and obtaining the first extract; 3) taking the residue after solid-liquid separation in step 2) and boiling it with water, separating the solid and liquid, collecting the extract and combining it with the first extract to obtain a total water extract; 4) taking Aster tataricus, Coltsfoot flos, and Pinellia ternata, soaking them in an ethanol solution, performing percolation, and collecting the percolation liquid; 5) mixing the total water extract and the percolate, and concentrating them to obtain an extract; 6) Soak the asarum in water and then distill it to extract the volatile oil; 7) mixing the extract with the volatile oil to obtain the Shegan Mahuang decoction extract.

2. The method for preparing the Shegan Mahuang Tang extract according to claim 1, characterized in that: The weight proportions of the raw materials for preparing the Shegan Mahuang decoction extract are: 7-10 parts of the Shegan, 10-14 parts of ephedra, 10-14 parts of schisandra, 10-14 parts of ginger, 20-30 parts of jujube, 7-10 parts of aster, 7-10 parts of coltsfoot, 10-14 parts of pinellia and 7-10 parts of asarum.

3. The method for preparing the Shegan Mahuang Tang extract according to claim 2, characterized in that: The amount of water used for soaking in step 1) is 8 to 10 times the total mass of Belamcanda chinensis, Schisandra chinensis, ginger and jujube, and the soaking time is 0.5 to 1 hour.

4. The method for preparing the Shegan Mahuang Tang extract according to claim 3, characterized in that: In step 2), the amount of water used for soaking is 15 to 20 times the mass of ephedra, the soaking time is 0.5 to 1 hour, the time for boiling separately is 10 to 20 minutes, and the time for boiling together is 0.5 to 1 hour.

5. The method for preparing the Shegan Mahuang Tang extract according to claim 4, characterized in that: The amount of water added in step 3) is equal to the sum of the amount of water used for soaking in steps 1) and 2), the decoction is performed 1 to 2 times, each time for 0.5 to 1 hour, and the extract and the residue are separated after each decoction, the residue is used for decoction again, and the extract is collected for later use.

6. The method for preparing the Shegan Mahuang Tang extract according to claim 5, characterized in that: The volume concentration of the ethanol solution in step 4) is 60-65%, the amount of ethanol used during the soaking is 3-5 times the total mass of Aster, Coltsfoot and Pinellia ternata, the filtration uses an ethanol solution with a volume concentration of 60-65% as an extractant, the amount used is 8-12 times the total mass of Aster, Coltsfoot and Pinellia ternata, the flow rate of the filtration is 0.7-1.2 mL / min, and the temperature is 15-25° C.

7. The method for preparing the Shegan Mahuang Tang extract according to claim 6, characterized in that: The amount of water used for soaking in step 6) is 15 to 20 times the mass of Asarum, the soaking time is 0.5 to 1 hour, and the distillation extraction time is 4 to 6 hours.

8. A Belamcanda Ephedra Decoction extract prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the Shegan Mahuang decoction extract according to claim 8 in the preparation of a medicine for treating bronchial asthma.

10. The use according to claim 9, characterized in that The drug is a liquid preparation or a solid preparation containing pharmaceutically acceptable excipients; The liquid preparation is a decoction, a suspension or an emulsion, and the solid preparation is a tablet, a capsule, a powder, a pill or a granule.

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