Pharmaceutical composition containing ambroxol and application of pharmaceutical composition in treating children asthma

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition containing ambroxol and application of the pharmaceutical composition to children asthma. The pharmaceutical composition containing ambroxol is prepared from the following components in parts by weight: 10 to 30 parts of ambroxol hydrochloride and 30 to 60 parts of fructus polygoni orientalis extract. The ambroxol hydrochloride and the fructus polygoni orientalis extract are combined to form the novel pharmaceutical composition, and animal experiments show that the pharmaceutical composition can remarkably reduce the airway sensitivity of rats and reduce the IL-4 and IgE levels in serum of the rats.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition containing ambroxol and application thereof in treating childhood asthma. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Asthma is a common chronic airway inflammatory disease in children, and its incidence is increasing worldwide. Childhood asthma not only seriously affects the quality of life of children, but may also have long-term adverse effects on their growth and development and lung function.

[0004] Western medicine plays an important role in the treatment of asthma. Ambroxol is a drug widely used in the treatment of respiratory diseases. It has the properties of promoting mucus discharge, dissolving sputum, and having certain anti-inflammatory properties. By regulating the secretion of respiratory mucus and promoting ciliary movement, ambroxol helps improve the airway patency of children with asthma, relieve symptoms such as coughing and sputum, and relieve airway inflammation to a certain extent. However, Western medicine treatment often has some limitations. For example, long-term use may lead to adverse reactions, and some children are not responsive enough to single Western medicine treatment, making it difficult to completely control asthma symptoms, especially in airway repair and prevention of recurrence after the acute attack of asthma. The effect needs to be further improved.

[0005] Traditional Chinese medicine has a long history and rich experience in the treatment of childhood asthma. Many Chinese herbal compound prescriptions or single Chinese medicines have been proven to have the effects of relieving asthma, relieving cough, resolving phlegm, anti-inflammatory and regulating the body's immune function. For example, some Chinese medicines can reduce airway inflammation by inhibiting the activation of inflammatory cells and the release of inflammatory mediators; some can regulate the body's immune balance, enhance the resistance of children, and reduce the recurrence rate of asthma. However, Chinese medicine treatment also faces some challenges, such as complex drug ingredients, unclear mechanism of action, difficulty in quality control, and relatively slow onset of effect.

[0006] In view of the respective characteristics and deficiencies of Western medicine and Chinese medicine in the treatment of childhood asthma, combining the two to form a new pharmaceutical composition has important research value and clinical significance. The combination of Chinese and Western medicine can give full play to the advantages of Western medicine in quickly relieving symptoms and Chinese medicine in overall regulation and improvement of body state, synergize each other, reduce the dosage of Western medicine and its adverse reactions, and make up for the defect of slow onset of Chinese medicine, improve the therapeutic effect and safety of childhood asthma, and provide a more comprehensive and effective drug option for the treatment of childhood asthma. Therefore, the development of a Chinese and Western medicine composition containing ambroxol for the treatment of childhood asthma has broad application prospects and urgent clinical needs. Summary of the invention

[0007] To overcome the deficiencies of the prior art, the present invention provides a Chinese and Western medicine composition containing ambroxol, wherein the ambroxol-containing medicine composition is composed of 10 to 30 parts by weight of ambroxol hydrochloride and 30 to 60 parts by weight of safflower fruit extract.

[0008] The preparation method of the safflower fruit extract is as follows:

[0009] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0010] (2) 10 g of safflower seed powder was placed in a container, 4 to 6 times of 60% to 80% ethanol containing 0.5 to 1 g of sodium butyrate was added, ultrasonicated for 45 to 90 min, filtered, centrifuged, and the supernatant was collected for later use;

[0011] (3) The supernatant is passed through a pre-treated AB-8 macroporous resin, the column is first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 30% to 50% ethanol solution, and the eluate is collected;

[0012] (4) Add 8 to 10 times of the mixed solvent to the residue of step (2), and add an appropriate amount of buffer solution, and perform vacuum rotary evaporation for 30 to 60 minutes, filter, mix the filtrate with the eluent, and concentrate it by vacuum rotary evaporation to a relative density of 1.35 to 1.45, and dry it to obtain a safflower fruit extract.

[0013] Furthermore, the buffer solution is selected from one of a phosphate buffer solution, a carbonate buffer solution, and a citrate buffer solution; the buffer solution is adjusted to pH = 4.5-5.8; the vacuum rotary evaporation temperature is 30°C-50°C, and the pressure is 10-30MPa; the mixed solvent is a mixed solvent of ethanol, acetone, and water in a volume ratio of 3-5:1-3:2-4; preferably, the mixed solvent is a mixed solvent of ethanol, acetone, and water in a volume ratio of 4:2:3.

[0014] The present invention also provides a preparation made from the above-mentioned pharmaceutical composition containing ambroxol and pharmaceutically acceptable adjuvants, including but not limited to tablets, granules, capsules and oral liquids.

[0015] The present invention also verifies the application of the above-mentioned pharmaceutical composition containing ambroxol in the preparation of a drug for treating asthma by using animal experiments.

[0016] Compared with the prior art, the technical effects of the present invention are:

[0017] The present invention makes up for the deficiency of western medicine in the treatment of asthma, combines ambroxol hydrochloride with safflower fruit extract to form a new pharmaceutical composition, and animal experiments show that the pharmaceutical composition of the present invention can significantly reduce the airway sensitivity of rats and reduce the IL-4 and IgE levels in rat serum. In addition, the present invention improves the preparation method of safflower fruit extract and increases the extraction rate of effective components. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : The contents of taxifolin and quercetin in the extracts of Carthamus tinctorius L. in Examples 1 to 3 and the extracts of Carthamus tinctorius L. in Comparative Examples 1 to 3.

[0019] Figure 2 :The results of airway responsiveness test in rats of each group

[0020] Figure 3 : IL-4 levels in serum of rats in each group.

[0021] Figure 4 : The levels of IgE in serum of rats in each group. DETAILED DESCRIPTION

[0022] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0023] Preparation and quality evaluation of extract from Fructus Polygoni Orientalis

[0024] Example 1: Extract of Polygonum multiflorum

[0025] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0026] (2) 10 g of safflower seed powder was placed in a container, 0.8 g of sodium butyrate was dissolved in 60% to 80% ethanol in 5 times the volume, ultrasonicated for 60 min, filtered, centrifuged, and the supernatant was collected for later use;

[0027] (3) The supernatant was passed through a pre-treated AB-8 macroporous resin, the column was first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 40% ethanol solution, and the eluate was collected;

[0028] (4) Add 9 times the volume ratio of 4:2:3 of ethanol, acetone and water to the residue of step (2), and add an appropriate amount of phosphate buffer solution to adjust the pH to 5.2, and perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 45 min, filter, mix the filtrate with the eluate, and concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.40, and dry to obtain a safflower fruit extract.

[0029] Example 2: Extract of safflower seeds

[0030] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0031] (2) 10 g of safflower seed powder was placed in a container, 0.5 g of sodium butyrate was dissolved in 4 times of 60% ethanol, ultrasonicated for 45 min, filtered, centrifuged, and the supernatant was collected for later use;

[0032] (3) The supernatant was passed through a pre-treated AB-8 macroporous resin, the column was first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 30% ethanol solution, and the eluate was collected;

[0033] (4) Add 8 times of a mixed solvent of ethanol, acetone and water in a volume ratio of 3:1:2 to the residue of step (2), and add an appropriate amount of carbonate buffer solution to adjust the pH to 5.8, and perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 30 min, filter, mix the filtrate with the eluent, and concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.35, and dry to obtain a safflower fruit extract.

[0034] Example 3: Extract of safflower seeds

[0035] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0036] (2) 10 g of safflower seed powder was placed in a container, 6 times of 80% ethanol containing 1 g of sodium butyrate was added, ultrasonicated for 90 min, filtered, centrifuged, and the supernatant was collected for later use;

[0037] (3) The supernatant is passed through a pre-treated AB-8 macroporous resin, the column is first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 50% ethanol solution, and the eluate is collected;

[0038] (4) Add 10 times of a mixed solvent of ethanol, acetone and water in a volume ratio of 5:3:4 to the residue of step (2), and add an appropriate amount of citrate buffer solution to adjust the pH to 4.5, perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 60 min, filter, mix the filtrate with the eluent, concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.45, and dry to obtain a safflower fruit extract.

[0039] Comparative Example 1: Extract of safflower seeds

[0040] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0041] (2) Take 10 g of safflower seed powder and place it in a container, add 5 times 60% to 80% ethanol, sonicate for 60 min, filter, centrifuge, and take the supernatant for later use;

[0042] (3) The supernatant was passed through a pre-treated AB-8 macroporous resin, the column was first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 40% ethanol solution, and the eluate was collected;

[0043] (4) Add 9 times the volume ratio of 4:2:3 of ethanol, acetone and water to the residue of step (2), and add an appropriate amount of phosphate buffer solution to adjust the pH to 5.2, and perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 45 min, filter, mix the filtrate with the eluate, and concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.40, and dry to obtain a safflower fruit extract.

[0044] Comparative Example 2: Extract of safflower seeds

[0045] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0046] (2) 10 g of safflower seed powder was placed in a container, 0.8 g of sodium acetate was dissolved in 60% to 80% ethanol in 5 times the volume, ultrasonicated for 60 min, filtered, centrifuged, and the supernatant was collected for later use;

[0047] (3) The supernatant was passed through a pre-treated AB-8 macroporous resin, the column was first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 40% ethanol solution, and the eluate was collected;

[0048] (4) Add 9 times the volume ratio of 4:2:3 of ethanol, acetone and water to the residue of step (2), and add an appropriate amount of phosphate buffer solution to adjust the pH to 5.2, and perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 45 min, filter, mix the filtrate with the eluate, and concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.40, and dry to obtain a safflower fruit extract.

[0049] Comparative Example 3: Extract of safflower seeds

[0050] (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder;

[0051] (2) 10 g of safflower seed powder was placed in a container, 0.8 g of sodium butyrate was dissolved in 60% to 80% ethanol in 5 times the volume, ultrasonicated for 60 min, filtered, centrifuged, and the supernatant was collected for later use;

[0052] (3) The supernatant was passed through a pre-treated AB-8 macroporous resin, the column was first rinsed with deionized water to remove water-soluble impurities, and then eluted with a 40% ethanol solution, and the eluate was collected;

[0053] (4) Add 9 times of a mixed solvent of ethanol, methanol and water in a volume ratio of 4:2:3 to the residue of step (2), and add an appropriate amount of phosphate buffer solution to adjust the pH to 5.2, and perform vacuum rotary evaporation at 30°C to 40°C (10 to 30 MPa) for 45 min, filter, mix the filtrate with the eluate, and concentrate at 40°C to 50°C (10 to 30 MPa) to a relative density of 1.40, and dry to obtain a safflower fruit extract.

[0054] The content of Taxus glabra extract was determined according to the high performance liquid chromatography method (General Rule 0512) of the 2020 edition of the Chinese Pharmacopoeia.

[0055] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; the detection wavelength is 290nm. The theoretical plate number calculated based on the Taxifolin peak should be no less than 6000.

[0056] Table 1 Elution program

[0057]

[0058] Preparation of reference solution: Take an appropriate amount of Taxol reference, weigh accurately, add methanol to make a solution containing 70μg per 1ml, and obtain. Preparation of test solution: Take about 0.5g of the powder of this product (passed through No. 3 sieve), weigh accurately, put it in a stoppered conical bottle, accurately add 25ml of methanol, weigh the weight, heat and reflux for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate, and obtain. Determination method: Accurately aspirate 10μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, and obtain.

[0059] Quercetin was determined according to the high performance liquid chromatography method (General Rule 0512) of the 2020 edition of the Chinese Pharmacopoeia.

[0060] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as filler; methanol-0.4% phosphoric acid solution (50:50) is used as mobile phase; detection wavelength is 360nm. The theoretical plate number calculated based on the quercetin peak should not be less than 2500. Preparation of reference solution: Take an appropriate amount of quercetin reference substance, accurately weigh it, and add 80% methanol to make a solution containing 20μg per 1ml. Preparation of test solution: Take about 1.5g of the powder of this product (passed through No. 3 sieve), weigh accurately, place in a stoppered conical bottle, accurately add 50ml of 80% methanol, weigh, heat and reflux for 1.5 hours, cool, weigh again, make up the lost weight with 80% methanol, shake well, filter, accurately measure 20ml of the filtrate, accurately add 7ml of 25% hydrochloric acid solution, place in a 85℃ water bath for hydrolysis for 30 minutes, take out, cool quickly, transfer to a 50ml volumetric bottle, add methanol to dilute to the scale, shake well, filter, take the filtrate, and get it. Determination method: Accurately aspirate 10μl of the reference solution and the test solution, inject into the liquid chromatograph, and determine, and get it.

[0061] Figure 1 The contents of taxifolin and quercetin in the extracts of the fruit of Polygonum multiflorum in Examples 1 to 3 and the extracts of the fruit of Polygonum multiflorum in Comparative Examples 1 to 3 are shown in the results. The results show that the present invention improves the preparation method of the fruit of Polygonum multiflorum extract, especially the addition of an appropriate amount of sodium butyrate-ethanol solution for extraction in step (2), which significantly increases the contents of taxifolin and quercetin, the active ingredients. It should be noted that in the screening experiment, sodium propionate-ethanol and sodium lactate-ethanol were also used to extract the fruit of Polygonum multiflorum powder, but the extraction effect of the active ingredients was far inferior to that of the sodium butyrate-ethanol solution, and even inferior to that of the sodium acetate-ethanol solution.

[0062] Composition tablet containing ambroxol

[0063] Each tablet contains: 20 mg of ambroxol hydrochloride, 45 mg of safflower fruit extract of Example 1, 100 mg of starch, 30 mg of microcrystalline cellulose, 5 mg of low-substituted hydroxypropyl methylcellulose, and 1 mg of magnesium stearate. The tablet is prepared by a powder tableting method.

[0064] Composition granules containing ambroxol

[0065] Each bag contains: 30 mg of ambroxol hydrochloride, 60 mg of safflower fruit extract of Example 2, 80 mg of starch, 20 mg of microcrystalline cellulose, 20 mg of mannitol, 30 mg of sucrose, 2 mg of sodium carboxymethyl starch, and 0.5 mg of micropowdered silica gel. The dry granulation method is used to prepare the product.

[0066] Composition capsule containing ambroxol

[0067] Each capsule contains: 10 mg of ambroxol hydrochloride, 30 mg of the extract of safflower seeds in Example 1, 50 mg of starch, 40 mg of lactose, and 1 mg of talc. The granules are prepared by dry method and filled into capsule shells.

[0068] Composition oral solution containing ambroxol

[0069] Each vial contains: 10 mg of ambroxol hydrochloride, 30 mg of safflower fruit extract of Example 1, 20 mg of polyethylene glycol, 0.5 mg of sodium benzoate, and 80 mg of sucrose. Add water to the full amount to obtain the product.

[0070] Pharmacodynamics experiments

[0071] The inventors conducted relevant pharmacodynamics experiments to prove the efficacy of the pharmaceutical composition containing ambroxol in treating asthma. It should be noted that the following experimental studies were conducted on the basis of the acute toxicity test and the long-term toxicity test to prove the safety of the drug, and the dosage in the experimental studies was within the safe dosage range.

[0072] Experimental animals: SD rats, SPF grade, 180-220 g, experimental animal license number: SYXK(Lu)20180008, adaptively fed under standard conditions for 1 week before the experiment.

[0073] Modeling method: The asthma model of rats was established by sensitization and stimulation with ovalbumin (OVA). On the 1st and 8th day of modeling, 1 ml of OVA sensitization solution was injected intraperitoneally for sensitization. The blank group rats (10 rats) were injected intraperitoneally with normal saline instead of OVA sensitization solution at the same time and the same dose. From the 15th day, except for the blank group, the rats were placed in the nebulizer box and stimulated by nebulization with 1% OVA nebulizer solution for 20 minutes per day for a total of 7 days. The blank group rats were nebulized with normal saline instead of OVA nebulizer solution, and the time and method were the same as those of the asthma model group rats. The rats with successful modeling were divided into model group, composition A group, composition B group, composition C group, positive drug group, and safflower fruit extract group, with 10 rats in each group.

[0074] Dosage:

[0075] Composition group A: 5.4 mg / kg of ambroxol hydrochloride + 12.15 mg / kg of the extract of safflower seeds in Example 1, administered by oral gavage once a day for 7 consecutive days.

[0076] Composition group B: 2.7 mg / kg of ambroxol hydrochloride + 8.1 mg / kg of the extract of Fructus Polygoni Orientalis of Example 2, administered by oral gavage once a day for 7 consecutive days.

[0077] Composition group C: 8.1 mg / kg of ambroxol hydrochloride + 16.2 mg / kg of the extract of safflower seeds of Example 3, administered by oral gavage once a day for 7 consecutive days.

[0078] Positive drug group: 8.1 mg / kg of ambroxol hydrochloride tablets (National Medicine Standard No. H19980016) were administered by oral gavage once a day for 7 consecutive days.

[0079] The extract group of safflower seeds: 16.2 mg / kg of safflower seeds extract of Example 1 was administered by oral gavage once a day for 7 consecutive days.

[0080] Airway responsiveness measurement:

[0081] Airway reactivity refers to the degree of airway contraction caused by various physical, chemical, drug or allergen stimuli. It is one of the important characteristics of asthma. The degree of airway reactivity is closely related to the severity of asthma. The principle is: when irritants act on the airways, the airway smooth muscles contract and the airway caliber becomes smaller, resulting in a decrease in ventilation function. The reactivity of the airways is reflected by measuring changes in ventilation function. Figure 2 The results of airway responsiveness test of rats in each group show that the airway sensitivity of rats in group A, group B and group C decreased after administration. There were significant differences between group A, group B and group C and the positive drug group (P<0.01).

[0082] Rat serum IL-4 and IgE content:

[0083] IL-4 (interleukin-4) plays a key role in the immune pathogenesis of asthma. It is mainly secreted by helper T cells 2 (Th2) cells and is a key cytokine that promotes B cells to produce IgE (immunoglobulin E). IL-4 can induce class switching of B cells, causing them to switch from producing other immunoglobulin types to producing IgE. Figure 3 The IL-4 content in the serum of rats in each group was significantly different between the composition group A, composition group B and composition group C and the positive drug group (P<0.01).

[0084] IgE is the core antibody that mediates allergic reactions in asthma. When asthma patients come into contact with allergens, the allergens cross-link with IgE bound to the surface of mast cells and basophils, activating these cells and causing them to release pre-synthesized inflammatory mediators, such as histamine and heparin, which can quickly cause bronchial smooth muscle contraction and increased vascular permeability, leading to acute asthma symptoms such as wheezing, coughing, and dyspnea. Figure 4 is the IgE content in the serum of rats in each group. Compared with the positive drug group, the difference between the composition group A, the composition group B and the composition group C was significant (P<0.01).

[0085] The above experiments used SPSS22.0 software to perform statistical analysis on the obtained data. The comparison among multiple groups was performed by one-way analysis of variance, and the comparison between two groups was performed by independent sample T test. P < 0.05 was considered statistically significant.

Claims

1. A pharmaceutical composition containing ambroxol, characterized in that, The ambroxol-containing pharmaceutical composition consists of 10 to 30 parts by weight of ambroxol hydrochloride and 30 to 60 parts by weight of safflower fruit extract.

2. The pharmaceutical composition containing ambroxol according to claim 1, characterized in that, The preparation method of the safflower fruit extract is as follows: (1) grinding the dried safflower seeds into fine powder and sieving to obtain safflower seeds powder; (2) 10 g of safflower seed powder was placed in a container, 4 to 6 times of 60% to 80% ethanol containing 0.5 to 1 g of sodium butyrate was added, ultrasonicated for 45 to 90 min, filtered, centrifuged, and the supernatant was collected for later use; (3) The supernatant is passed through a pre-treated AB-8 macroporous resin, firstly rinsed with deionized water to remove water-soluble impurities, then eluted with 30% to 50% ethanol solution, and the eluate is collected; (4) Add 8 to 10 times of the mixed solvent to the residue of step (2), and add an appropriate amount of buffer solution, and perform vacuum rotary evaporation for 30 to 60 minutes, filter, mix the filtrate with the eluent, and concentrate it by vacuum rotary evaporation to a relative density of 1.35 to 1.45, and dry it to obtain a safflower fruit extract.

3. The pharmaceutical composition containing ambroxol according to claim 2, characterized in that, The buffer solution is selected from one of a phosphate buffer solution, a carbonate buffer solution and a citrate buffer solution.

4. The pharmaceutical composition containing ambroxol according to claim 2, characterized in that, The buffer solution is adjusted to pH = 4.5-5.

8.

5. The pharmaceutical composition containing ambroxol according to claim 2, characterized in that, The vacuum rotary evaporation temperature is 30°C to 50°C.

6. The pharmaceutical composition containing ambroxol according to claim 2, characterized in that, The pressure of the reduced pressure rotary evaporation is 10-30 MPa.

7. The pharmaceutical composition containing ambroxol according to claim 2, characterized in that, The mixed solvent is a mixed solvent of ethanol, acetone and water in a volume ratio of 3 to 5:1 to 3:2 to 4; preferably, the mixed solvent is a mixed solvent of ethanol, acetone and water in a volume ratio of 4:2:

3.

8. Use of the pharmaceutical composition containing ambroxol according to claim 1 in preparing a drug for treating childhood asthma.

9. A preparation, characterized in that The preparation is made of the pharmaceutical composition containing ambroxol according to claim 1 and pharmaceutically acceptable excipients.

10. The preparation according to claim 9, characterized in that The preparation is in the form of tablets, granules, capsules and oral liquid.

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