A Chinese medicine composition for treating allergic rhinitis and its preparation method and application

Abstract

The present invention proposes a Chinese medicine composition for treating allergic rhinitis, and a preparation method and application thereof, which belong to the field of pharmaceutical technology. Ag-graphene oxide is adsorbed on in vitro proliferated γδT cells, a Chinese medicine fermentation product obtained by fermentation and extraction of Xanthium sibiricum, Astragalus, Poria, Magnolia officinalis, and Mentha is prepared, berberine and quercetin are prepared into eutectic powder, the three are mixed and added into water, probiotics, carboxymethyl chitosan, poloxamer 407 and poloxamer 188 are added, and mixed evenly to prepare a Chinese medicine composition for treating allergic rhinitis, which has a good effect of improving allergic rhinitis, adopts a method of combining traditional Chinese and Western medicine, reduces inflammatory response, enhances the immune function of patients, improves the clinical symptoms of patients with allergic rhinitis, reduces the long-term recurrence rate, reduces the use of drugs, improves the quality of life, and reduces the risk of developing asthma, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of medicines, and in particular to a Chinese medicine composition for treating allergic rhinitis, and a preparation method and application thereof. Background Art

[0002] Rhinitis is a common clinical disease, which is an inflammation of the nasal mucosa caused by viruses, bacteria, allergens, various physical and chemical factors, and certain systemic diseases. Allergic rhinitis refers to a non-infectious inflammatory disease of the nasal mucosa, which is mainly caused by the release of IgE-mediated mediators (mainly histamine) after atopic individuals come into contact with allergens, and involves the participation of a variety of immune active cells and cytokines. The typical symptoms of allergic rhinitis are mainly paroxysmal sneezing, clear nasal discharge, nasal congestion and nasal itching, and some are accompanied by hyposmia.

[0003] At present, the following methods are mainly used to treat allergic rhinitis: (1) avoid contact with allergens; (2) drug treatment; (3) immunotherapy; (4) surgical treatment. Among them, drug treatment is currently an important treatment method commonly used, and most of the commonly used drugs are Western medicines, such as antihistamines, glucocorticoids, anti-leukotriene drugs, etc. However, these drugs have certain toxic side effects on the human body and still cannot be completely cured. Traditional Chinese medicine therapy has the advantages of safety, reliability, few side effects, sustained efficacy, and reduced recurrence. Therefore, it is very necessary to further explore drugs for the treatment of allergic rhinitis in the field of traditional Chinese medicine.

[0004] Chinese invention patent application CN117357575A discloses a Chinese medicine composition for external treatment of rhinitis and its preparation method, which comprises, by weight: 5-15g of magnolia, 12-24g of schizonepeta, 12-24g of siler, 20-30g of atractylodes, 4-12g of angelica, 3-9g of cicada shell, 20-30g of kudzu root and 12-24g of sophora flavescens. However, the active components of the Chinese medicine composition are obtained by simply extracting the Chinese medicine raw materials with water, and the efficacy is not obvious. Summary of the invention

[0005] The purpose of the present invention is to propose a Chinese medicine composition for treating allergic rhinitis, a preparation method and an application thereof, which has a good effect of improving allergic rhinitis, adopts a method of combining Chinese and Western medicine to reduce inflammatory response, enhance the patient's immune function, improve the clinical symptoms of patients with allergic rhinitis, reduce the long-term recurrence rate, reduce the use of drugs, improve the quality of life, and reduce the risk of developing asthma, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a method for preparing a traditional Chinese medicine composition for treating allergic rhinitis. The method comprises the following steps: adsorbing in vitro proliferated γδT cells onto Ag-graphene oxide to prepare a traditional Chinese medicine fermentation product obtained by fermenting and extracting Xanthium sibiricum, Astragalus membranaceus, Poria cocos, Flos magnoliae and Mentha arvensis; preparing eutectic powder from berberine and quercetin; mixing the three and adding them into water; adding probiotics, carboxymethyl chitosan, poloxamer 407 and poloxamer 188, and mixing them uniformly to prepare the traditional Chinese medicine composition for treating allergic rhinitis.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Isolation and extraction of γδT cells: Anticoagulate peripheral blood, separate mononuclear cells by density gradient centrifugation with lymphocyte separation medium, add culture medium, induce proliferation, centrifuge, collect cell pellets, wash with physiological saline, and obtain γδT cells;

[0010] S2. Preparation of Ag-graphene oxide particles that adsorb γδT cells: adding silver nitrate and glucose to a graphene oxide aqueous dispersion, heating and stirring to react, dialyzing, and freeze-drying to obtain Ag-graphene oxide powder, adding the Ag-graphene oxide powder to physiological saline, adding the γδT cells obtained in step S1, stirring and mixing evenly, and freeze-drying to obtain Ag-graphene oxide particles that adsorb γδT cells;

[0011] S3. Preparation of Chinese medicine fermentation products: Xanthium sibiricum, Astragalus, Poria, Magnolia, and mint were washed, dried, crushed, added to water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, fermented, filtered, washed with solids, concentrated to obtain a concentrated bacterial solution, and the filtrate was freeze-dried to obtain a Chinese medicine fermentation product;

[0012] S4. Preparation of eutectic powder: dissolving berberine in chloroform, dissolving quercetin in dimethyl sulfoxide, and dissolving matrine in ethanol, mixing the three solutions evenly, evaporating the solvent at room temperature, filtering, washing, ball milling, and drying to obtain eutectic powder;

[0013] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: uniformly mix the Ag-graphene oxide particles adsorbing γδT cells prepared in step S2, the Chinese medicine fermentation product prepared in step S3, the concentrated bacterial liquid prepared in step S3, and the eutectic powder prepared in step S4, add water, carboxymethyl chitosan, poloxamer 407 and poloxamer 188, mix uniformly, and swell to prepare a Chinese medicine composition for treating allergic rhinitis.

[0014] As a further improvement of the present invention, the anticoagulation treatment in step S1 is to add 1-2wt% sodium heparin to the peripheral blood, the culture medium is 1640 culture medium containing 2-4mmol / L glutamine and 10-15mg / L zoledronic acid, the mass ratio of the mononuclear cells to the culture medium is 10-15:100, and the proliferation conditions are 36-38°C, 4-6% CO 2 Culture under the same conditions for 2-4 days.

[0015] As a further improvement of the present invention, the concentration of the graphene oxide aqueous dispersion in step S2 is 0.5-1 mg / mL, the mass ratio of the graphene oxide aqueous dispersion, silver nitrate and glucose is 100:2-4:3-5, the temperature of the heating and stirring reaction is 85-95°C, the time is 0.5-1h, and the mass ratio of the Ag-graphene oxide powder, γδT cells and physiological saline is 5-7:3-5:100.

[0016] As a further improvement of the present invention, the mass ratio of Xanthium sibiricum, Astragalus, Poria, Magnolia officinalis, Mint and water in step S3 is 3-5:2-4:2-4:1-2:0.5-1:100, the inoculation amount of Lactobacillus plantarum and Bifidobacterium infantis seed solution is 1-2v / v% and 2-3v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, the fermentation culture conditions are anaerobic conditions, 100-150r / min, 36-38°C, fermentation culture for 36-48h, and the bacterial content of the concentrated bacterial solution is 10 11 -10 12 cfu / mL.

[0017] As a further improvement of the present invention, in step S4, the mass ratio of berberine, quercetin and matrine is 1-2:3-5:2-4, the time for evaporating the solvent is 2-4 days, and the time for ball milling is 1-2 hours.

[0018] As a further improvement of the present invention, the mass ratio of the Ag-graphene oxide particles that adsorb γδT cells, the traditional Chinese medicine fermentation product, the concentrated bacterial liquid, the eutectic powder, water, carboxymethyl chitosan, poloxamer 407 and poloxamer 188 in step S5 is 3-5:15-20:10-20:0.5-1:100:12-15:2-3:1-2, the expansion temperature is 2-4°C, and the time is 18-24h.

[0019] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0020] S1. Isolation and extraction of γδT cells: 1-2wt% sodium heparin was added to the peripheral blood for anticoagulation, and mononuclear cells were separated by density gradient centrifugation of lymphocyte separation fluid, and culture medium was added. The mass ratio of mononuclear cells to culture medium was 10-15:100, 36-38°C, 4-6% CO 2 Induce proliferation and culture under the conditions for 2-4 days, centrifuge, take the cell pellet, wash with physiological saline, and obtain γδT cells;

[0021] The culture medium is a 1640 culture medium containing 2-4 mmol / L glutamine and 10-15 mg / L zoledronic acid;

[0022] S2. Preparation of Ag-graphene oxide particles adsorbing γδT cells: add 2-4 parts by weight of silver nitrate and 3-5 parts by weight of glucose to 100 parts by weight of graphene oxide aqueous dispersion, heat to 85-95°C, stir to react for 0.5-1h, dialyze, freeze-dry to obtain Ag-graphene oxide powder, add 5-7 parts by weight of Ag-graphene oxide powder to 100 parts by weight of physiological saline, add 3-5 parts by weight of γδT cells obtained in step S1, stir to mix evenly, freeze-dry to obtain Ag-graphene oxide particles adsorbing γδT cells;

[0023] S3. Preparation of Chinese medicine fermentation products: 3-5 parts by weight of Xanthium sibiricum, 2-4 parts by weight of Astragalus, 2-4 parts by weight of Poria, 1-2 parts by weight of Magnolia, 0.5-1 parts by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amounts were 1-2v / v% and 2-3v / v%, respectively, the seed solution containing bacteria was 10 8 -10 9 cfu / mL, under anaerobic conditions, 100-150r / min, 36-38°C, fermentation culture for 36-48h, filtration, solid washing, concentration to obtain concentrated bacterial solution, lyophilization of the filtrate, and preparation of Chinese medicine fermentation products;

[0024] S4. Preparation of eutectic powder: 1-2 parts by weight of berberine was dissolved in 50 parts by weight of chloroform, 3-5 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 2-4 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed uniformly, the solvent was evaporated at room temperature for 2-4d, filtered, washed, ball milled for 1-2h, and dried to obtain eutectic powder;

[0025] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 3-5 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 15-20 parts by weight of the Chinese medicine fermentation product prepared in step S3, 10-20 parts by weight of the concentrated bacterial solution prepared in step S3, and 0.5-1 parts by weight of the eutectic powder prepared in step S4 are mixed evenly, 100 parts by weight of water, 12-15 parts by weight of carboxymethyl chitosan, 2-3 parts by weight of poloxamer 407 and 1-2 parts by weight of poloxamer 188 are added, mixed evenly, and expanded at 2-4°C for 18-24 hours to prepare a Chinese medicine composition for treating allergic rhinitis.

[0026] The present invention further protects a traditional Chinese medicine composition for treating allergic rhinitis prepared by the above preparation method.

[0027] The present invention further protects the use of the above-mentioned traditional Chinese medicine composition for treating allergic rhinitis in preparing medicines for alleviating, improving and treating allergic rhinitis.

[0028] The present invention has the following beneficial effects:

[0029] Allergic rhinitis (AR) is an IgE-mediated allergic disease characterized by helper T cell dysfunction. It can cause patients to experience symptoms such as nasal congestion, nasal itching, paroxysmal sneezing, and clear nasal discharge. The purpose of treatment is to alleviate allergic symptoms, reduce drug use, improve quality of life, and reduce the risk of developing asthma.

[0030] Western medicine can temporarily relieve symptoms after taking it, but it has the disadvantages of easy dependence, drug resistance and adverse reactions. Chinese and Western medicine can treat allergic rhinitis not only to clear the orifices and remove turbidity, but also to relieve the symptoms of heat stagnation in the gallbladder meridian. Western medicine can significantly improve the symptoms, while Chinese medicine has good anti-inflammatory, anti-allergic and anti-edema effects, is safer, will not cause drug dependence, has almost no adverse reactions, and has a synergistic effect.

[0031] Berberine, also known as berberine, is an alkaline substance extracted from the Chinese medicine Coptis chinensis. It has strong antibacterial and anti-inflammatory activity and can relieve symptoms, but it cannot change the overall immune level of the patient, and berberine is insoluble in water, which greatly limits its application. Quercetin is a flavonoid monomer compound widely present in plants, with antioxidant and anti-inflammatory activity, mainly manifested as the inhibitory effect of inflammatory factors, but quercetin is also insoluble in water, so its bioavailability is poor. Matrine is an alkaloid, and its main pharmacological effects include inhibiting histamine release, anti-inflammatory, and anti-allergic. Mixing berberine, quercetin and matrine to prepare cocrystals greatly improves the solubility of each component in water and its bioavailability. By improving the water solubility of the drug cocrystal, it is easier to adhere to the nasal mucosa, the residence time is increased, and it is not easy to be removed by cilia, which increases the onset time. At the same time, the cocrystal powder has good antibacterial, antioxidant, and anti-allergic effects, and has a good therapeutic and relieving effect on allergic rhinitis.

[0032] In the present invention, in the Chinese medicine composition, Xanthium sibiricum is sweet and warm, mainly dispersing wind and opening the orifices; Astragalus is sweet and slightly warm, Poria is sweet, replenishing qi and raising yang, penetrating dampness, strengthening the spleen, calming the mind and tranquilizing the nerves, and has a good effect of improving immunity; Magnolia lily is pungent and warm, dispersing wind evil, and clearing the nasal orifices; Peppermint is pungent and cool, mainly dispersing wind-heat, and clearing the head. All the medicines can go up into the lungs to the top of the head, dispersing wind evil, promoting lung qi, clearing the nasal orifices, warming the lungs and dispersing cold, strengthening the spleen and replenishing qi, warming the kidneys and strengthening yang, thereby having the effects of antibacterial and anti-inflammatory, enhancing immunity, inhibiting sweat and antibacterial, replenishing qi in the middle position, and preventing allergies.

[0033] The culture medium used in the γδT cells prepared in vitro of the present invention contains glutamine and zoledronic acid, which can well improve the resistance of the γδT cells, induce their proliferation, and improve their in vitro stability.

[0034] The present invention prepares a kind of Ag-graphene oxide particles for adsorbing γδT cells, and obtains Ag-graphene oxide composites by in-situ reduction of graphene oxide, which has good antibacterial properties. Meanwhile, graphene oxide is rich in hydroxyl groups, which is convenient for adsorbing and fixing γδT cells. Ag-graphene oxide has good electron transfer ability, and local relief of nose inflammation by radiation heating electron transfer improves rhinitis symptoms, and promotes the release of γδT cells at the same time. γδT cells can regulate Treg cells to correct IL-17+γδT cell immune imbalance to treat allergic rhinitis, and can induce the generation of Th1 and Th2 related factors, and interact with nasal mucosal epithelial cells to improve the symptoms of inflammation. In the process of radiation heating, IL-4 and gamma interferon secreted by γδT cells can also be promoted, inflammatory response can be alleviated, the immune function of patients can be enhanced, the clinical symptoms of patients with allergic rhinitis can be improved, and the long-term recurrence rate can be reduced.

[0035] In addition, probiotics are added to the composition of the present invention to reduce the symptoms of rhinitis caused by dust mites and pathogenic bacteria, reduce the occurrence of symptom-induced allergic rhinitis, and thus improve the symptoms of allergic rhinitis.

[0036] The Chinese medicine composition for treating allergic rhinitis prepared by the present invention is a thermosensitive hydrogel, which is in a solution state at room temperature and is converted into a gel state after being sprayed into the nasal cavity, thereby greatly improving the sustained release effect of the drug, prolonging the action time of the drug, and further improving the efficacy of the drug.

[0037] The Chinese medicine composition for treating allergic rhinitis prepared by the present invention has a good effect of improving allergic rhinitis, adopts a method of combining traditional Chinese and western medicine to reduce inflammatory response, enhance the patient's immune function, improve the clinical symptoms of patients with allergic rhinitis, reduce the long-term recurrence rate, reduce the use of drugs, improve the quality of life, and reduce the risk of developing asthma, and has broad application prospects. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Human lymphocyte separation medium was purchased from GE.

[0040] Graphene oxide aqueous dispersion, 1 mg / mL, was purchased from Zhejiang Zhiti Nano Micro New Materials Co., Ltd.

[0041] Lactobacillus plantarum, 10 billion cfu / g, and Bifidobacterium infantis, 10 billion cfu / g, were purchased from Jiangsu Caiwei Biotechnology Co., Ltd.

[0042] Preparation method of Lactobacillus plantarum and Bifidobacterium infantis seed solution: inoculate the strains into a slant culture medium, culture at 37°C in anoxic environment for 18 hours, and obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0043] Example 1

[0044] This embodiment provides a method for preparing a Chinese medicine composition for treating allergic rhinitis, which specifically comprises the following steps:

[0045] S1. Isolation and extraction of γδT cells: 1 wt% sodium heparin was added to human peripheral blood for anticoagulation, and mononuclear cells were separated by density gradient centrifugation of human lymphocyte separation medium. Culture medium was added, and the mass ratio of mononuclear cells to culture medium was 10:100. The culture medium was incubated at 36°C and 4% CO.2 The proliferation was induced under the conditions for 4 days, and the cells were centrifuged, the cell pellets were collected, and washed with physiological saline to obtain γδT cells;

[0046] The culture medium is 1640 culture medium containing 2 mmol / L glutamine and 10 mg / L zoledronic acid;

[0047] S2. Preparation of Ag-graphene oxide particles for adsorbing γδT cells: Add 2 parts by weight of silver nitrate and 3 parts by weight of glucose to 100 parts by weight of 1 mg / mL graphene oxide aqueous dispersion, heat to 85°C, stir and react for 0.5h, dialyze to remove unreacted silver nitrate and glucose, freeze-dry to obtain Ag-graphene oxide powder, add 5 parts by weight of Ag-graphene oxide powder to 100 parts by weight of physiological saline, add 3 parts by weight of γδT cells obtained in step S1, stir and mix evenly, freeze-dry to obtain Ag-graphene oxide particles for adsorbing γδT cells;

[0048] S3. Preparation of Chinese medicine fermentation products: 3 parts by weight of Xanthium sibiricum, 2 parts by weight of Astragalus, 2 parts by weight of Poria, 1 part by weight of Magnolia, and 0.5 parts by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amount was 1v / v% and 2v / v%, respectively, under anaerobic conditions, 100r / min, 36°C, fermented and cultured for 36h, filtered, washed with solids, concentrated to obtain a concentrated bacterial solution, and the filtrate was freeze-dried to obtain a Chinese medicine fermentation product;

[0049] S4. Preparation of eutectic powder: 1 part by weight of berberine was dissolved in 50 parts by weight of chloroform, 3 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 2 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed uniformly, the solvent was evaporated at room temperature for 4d, filtered, washed, ball-milled for 1h, and dried to obtain eutectic powder;

[0050] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 3 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 15 parts by weight of the Chinese medicine fermentation product prepared in step S3, 10 parts by weight of the concentrated bacterial liquid prepared in step S3, and 0.5 parts by weight of the eutectic powder prepared in step S4 are mixed for 30 minutes, 100 parts by weight of water, 12 parts by weight of carboxymethyl chitosan, 2 parts by weight of poloxamer 407 and 1 part by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 2°C for 18 hours to obtain a Chinese medicine composition for treating allergic rhinitis.

[0051] Example 2

[0052] This embodiment provides a method for preparing a Chinese medicine composition for treating allergic rhinitis, which specifically comprises the following steps:

[0053] S1. Isolation and extraction of γδT cells: 2 wt% sodium heparin was added to human peripheral blood for anticoagulation, and mononuclear cells were separated by density gradient centrifugation of human lymphocyte separation medium. Culture medium was added, and the mass ratio of mononuclear cells to culture medium was 15:100. The culture medium was incubated at 38°C and 6% CO. 2 The proliferation was induced under the conditions for 4 days, and the cells were centrifuged, the cell pellets were collected, and washed with physiological saline to obtain γδT cells;

[0054] The culture medium is 1640 culture medium containing 4 mmol / L glutamine and 15 mg / L zoledronic acid;

[0055] S2. Preparation of Ag-graphene oxide particles for adsorbing γδT cells: Add 4 parts by weight of silver nitrate and 5 parts by weight of glucose to 100 parts by weight of 1 mg / mL graphene oxide aqueous dispersion, heat to 95° C., stir and react for 1 hour, dialyze to remove unreacted silver nitrate and glucose, freeze-dry to obtain Ag-graphene oxide powder, add 7 parts by weight of Ag-graphene oxide powder to 100 parts by weight of physiological saline, add 5 parts by weight of γδT cells obtained in step S1, stir and mix evenly, freeze-dry to obtain Ag-graphene oxide particles for adsorbing γδT cells;

[0056] S3. Preparation of Chinese medicine fermentation products: 5 parts by weight of Xanthium sibiricum, 4 parts by weight of Astragalus, 4 parts by weight of Poria, 2 parts by weight of Magnolia, and 1 part by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amount was 2v / v% and 3v / v%, respectively, under anaerobic conditions, 150r / min, 38°C, fermented and cultured for 48h, filtered, washed with solids, concentrated to obtain a concentrated bacterial solution, and the filtrate was freeze-dried to obtain a Chinese medicine fermentation product;

[0057] S4. Preparation of eutectic powder: 2 parts by weight of berberine was dissolved in 50 parts by weight of chloroform, 5 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 4 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed uniformly, the solvent was evaporated at room temperature for 2d, filtered, washed, ball-milled for 2h, and dried to obtain eutectic powder;

[0058] S5. Preparation of a traditional Chinese medicine composition for treating allergic rhinitis: 5 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 20 parts by weight of the traditional Chinese medicine fermentation product prepared in step S3, 20 parts by weight of the concentrated bacterial liquid prepared in step S3, and 1 part by weight of the eutectic powder prepared in step S4 are mixed for 30 minutes, 100 parts by weight of water, 15 parts by weight of carboxymethyl chitosan, 3 parts by weight of poloxamer 407 and 2 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 4°C for 24 hours to prepare a traditional Chinese medicine composition for treating allergic rhinitis.

[0059] Example 3

[0060] This embodiment provides a method for preparing a Chinese medicine composition for treating allergic rhinitis, which specifically comprises the following steps:

[0061] S1. Isolation and extraction of γδT cells: 1.5 wt% sodium heparin was added to human peripheral blood for anticoagulation, mononuclear cells were separated by density gradient centrifugation of human lymphocyte separation medium, and culture medium was added. The mass ratio of mononuclear cells to culture medium was 12:100, and the culture medium was incubated at 37°C and 5% CO. 2 The proliferation was induced under the conditions for 3 days, and the cells were centrifuged, the cell pellets were collected, and washed with physiological saline to obtain γδT cells;

[0062] The culture medium is 1640 culture medium containing 3 mmol / L glutamine and 12 mg / L zoledronic acid;

[0063] S2. Preparation of Ag-graphene oxide particles for adsorbing γδT cells: Add 3 parts by weight of silver nitrate and 4 parts by weight of glucose to 100 parts by weight of 1 mg / mL graphene oxide aqueous dispersion, heat to 90°C, stir and react for 1 hour, dialyze to remove unreacted silver nitrate and glucose, freeze-dry to obtain Ag-graphene oxide powder, add 6 parts by weight of Ag-graphene oxide powder to 100 parts by weight of physiological saline, add 4 parts by weight of γδT cells obtained in step S1, stir and mix evenly, freeze-dry to obtain Ag-graphene oxide particles for adsorbing γδT cells;

[0064] S3. Preparation of Chinese medicine fermentation products: 4 parts by weight of Xanthium sibiricum, 3 parts by weight of Astragalus, 3 parts by weight of Poria, 1.5 parts by weight of Magnolia, 0.7 parts by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amount was 1.5v / v% and 2.5v / v%, respectively, under anaerobic conditions, 120r / min, 37°C, fermented and cultured for 42h, filtered, washed with solids, concentrated to obtain a concentrated bacterial solution, and the filtrate was freeze-dried to obtain a Chinese medicine fermentation product;

[0065] S4. Preparation of eutectic powder: 1.5 parts by weight of berberine was dissolved in 50 parts by weight of chloroform, 4 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 3 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed uniformly, the solvent was evaporated at room temperature for 3d, filtered, washed, ball milled for 1.5h, and dried to obtain eutectic powder;

[0066] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 4 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 17 parts by weight of the Chinese medicine fermentation product prepared in step S3, 15 parts by weight of the concentrated bacterial liquid prepared in step S3, and 0.7 parts by weight of the eutectic powder prepared in step S4 are mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 3°C ​​for 21 hours to prepare a Chinese medicine composition for treating allergic rhinitis.

[0067] Example 4

[0068] Compared with Example 3, the difference is that Lactobacillus plantarum is not inoculated in step S3, and the inoculation amount of Bifidobacterium infantis seed liquid is 4 v / v%.

[0069] Example 5

[0070] Compared with Example 3, the difference is that in step S3, Bifidobacterium infantis is not inoculated, and the inoculation amount of Lactobacillus plantarum seed liquid is 4 v / v%.

[0071] Example 6

[0072] Compared with Example 3, the difference is that in step S1, glutamine in the culture medium is replaced by an equal mass of zoledronic acid.

[0073] Example 7

[0074] Compared with Example 3, the difference is that in step S1, zoledronic acid is not added to the culture medium but is replaced by an equal mass of glutamine.

[0075] Comparative Example 1

[0076] Compared with Example 3, the difference is that the culture medium in step S1 is 1640 culture medium.

[0077] Comparative Example 2

[0078] Compared with Example 3, the difference is that silver nitrate is not added in step S2.

[0079] The details are as follows:

[0080] S2. Preparation of graphene oxide-coated γδT cells: Add 6 parts by weight of graphene oxide powder to 100 parts by weight of normal saline, add 4 parts by weight of γδT cells prepared in step S1, stir and mix evenly, and freeze-dry to obtain graphene oxide-coated γδT cells.

[0081] Comparative Example 3

[0082] Compared with Example 3, the difference is that step S2 is not performed.

[0083] The details are as follows:

[0084] S1. Isolation and extraction of γδT cells: 1.5 wt% sodium heparin was added to human peripheral blood for anticoagulation, mononuclear cells were separated by density gradient centrifugation of human lymphocyte separation medium, and culture medium was added. The mass ratio of mononuclear cells to culture medium was 12:100, and the culture medium was incubated at 37°C and 5% CO. 2 The proliferation was induced under the conditions for 3 days, and the cells were centrifuged, the cell pellets were collected, and washed with physiological saline to obtain γδT cells;

[0085] The culture medium is 1640 culture medium containing 3 mmol / L glutamine and 12 mg / L zoledronic acid;

[0086] S2. Preparation of Chinese medicine fermentation products: 4 parts by weight of Xanthium sibiricum, 3 parts by weight of Astragalus, 3 parts by weight of Poria, 1.5 parts by weight of Magnolia, 0.7 parts by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amount was 1.5v / v% and 2.5v / v%, respectively, under anaerobic conditions, 120r / min, 37°C, fermented and cultured for 42h, filtered, washed with solids, concentrated to obtain a concentrated bacterial solution, and the filtrate was freeze-dried to obtain a Chinese medicine fermentation product;

[0087] S3. Preparation of eutectic powder: 1.5 parts by weight of berberine was dissolved in 50 parts by weight of chloroform, 4 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 3 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed, the solvent was evaporated at room temperature for 3d, filtered, washed, ball milled for 1.5h, and dried to obtain eutectic powder;

[0088] S4. Preparation of a Chinese medicine composition for treating allergic rhinitis: 4 parts by weight of γδT cells prepared in step S1, 17 parts by weight of the Chinese medicine fermentation product prepared in step S2, 15 parts by weight of the concentrated bacterial solution prepared in step S2, and 0.7 parts by weight of the eutectic powder prepared in step S3 are mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 3°C ​​for 21 hours to obtain a Chinese medicine composition for treating allergic rhinitis.

[0089] Comparative Example 4

[0090] Compared with Example 3, the difference is that fermentation is not performed in step S3, and heating to boiling extraction is used for 4 hours.

[0091] The details are as follows:

[0092] S3. Preparation of Chinese herbal medicine extracts: 4 parts by weight of Xanthium sibiricum, 3 parts by weight of Astragalus, 3 parts by weight of Poria, 1.5 parts by weight of Magnolia officinalis, and 0.7 parts by weight of Mentha were washed, dried, and crushed, added to 100 parts by weight of water, heated to boiling and extracted for 4 hours, filtered, and the filtrate was freeze-dried to obtain a Chinese herbal medicine extract.

[0093] Comparative Example 5

[0094] Compared with Example 3, the difference is that in step S4, berberine, quercetin and matrine are simply mixed.

[0095] The details are as follows:

[0096] S4. Preparation of a mixture: 2 parts by weight of berberine, 5 parts by weight of quercetin, and 4 parts by weight of matrine were mixed and ball-milled for 2 h, and dried to obtain a mixture.

[0097] Comparative Example 6

[0098] Compared with Example 3, the difference is that Ag-graphene oxide particles for adsorbing γδT cells are not added in step S5.

[0099] The details are as follows:

[0100] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 17 parts by weight of the Chinese medicine fermentation product prepared in step S3, 15 parts by weight of the concentrated bacterial liquid prepared in step S3, and 0.7 parts by weight of the eutectic powder prepared in step S4 were mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 were added, the mixture was stirred for 15 minutes, and expanded at 3°C ​​for 21 hours to obtain a Chinese medicine composition for treating allergic rhinitis.

[0101] Comparative Example 7

[0102] Compared with Example 3, the difference is that no Chinese medicine fermentation product is added in step S5.

[0103] The details are as follows:

[0104] S5. Preparation of a traditional Chinese medicine composition for treating allergic rhinitis: 4 parts by weight of the Ag-graphene oxide particles adsorbing γδT cells obtained in step S2, 15 parts by weight of the concentrated bacterial solution obtained in step S3, and 0.7 parts by weight of the eutectic powder obtained in step S4 are mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 3°C ​​for 21 hours to obtain a traditional Chinese medicine composition for treating allergic rhinitis.

[0105] Comparative Example 8

[0106] Compared with Example 3, the difference is that no concentrated bacterial solution is added in step S5.

[0107] The details are as follows:

[0108] S5. Preparation of a traditional Chinese medicine composition for treating allergic rhinitis: 4 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 17 parts by weight of the traditional Chinese medicine fermentation product prepared in step S3, and 0.7 parts by weight of the eutectic powder prepared in step S4 are mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 3°C ​​for 21 hours to obtain a traditional Chinese medicine composition for treating allergic rhinitis.

[0109] Comparative Example 9

[0110] Compared with Example 3, the difference is that no eutectic powder is added in step S5.

[0111] The details are as follows:

[0112] S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 4 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 17 parts by weight of the Chinese medicine fermentation product prepared in step S3, and 15 parts by weight of the concentrated bacterial solution prepared in step S3 are mixed for 30 minutes, 100 parts by weight of water, 13 parts by weight of carboxymethyl chitosan, 2.5 parts by weight of poloxamer 407 and 1.5 parts by weight of poloxamer 188 are added, the mixture is stirred for 15 minutes, and the mixture is expanded at 3°C ​​for 21 hours to prepare a Chinese medicine composition for treating allergic rhinitis.

[0113] Comparative Example 10

[0114] Compared with Example 3, the difference is that carboxymethyl chitosan, poloxamer 188 and poloxamer 407 are not added in step S5.

[0115] The details are as follows:

[0116] S5. Preparation of a traditional Chinese medicine composition for treating allergic rhinitis: Mix 4 parts by weight of the γδT cell-adsorbing Ag-graphene oxide particles prepared in step S2, 17 parts by weight of the traditional Chinese medicine fermentation product prepared in step S3, 15 parts by weight of the concentrated bacterial liquid prepared in step S3, and 0.7 parts by weight of the eutectic powder prepared in step S4 for 30 minutes, add 100 parts by weight of water, stir and mix for 15 minutes, and expand at 3°C ​​for 21 hours to obtain a traditional Chinese medicine composition for treating allergic rhinitis.

[0117] Test Example 1

[0118] Except for 10 healthy rats set as the healthy group, the rest of the rats used the ovalbumin sensitization method to establish an allergic rhinitis model. During the modeling process, 0.3 mg ovalbumin, 300 mg aluminum hydroxide powder and 1 ml normal saline were mixed to form a suspension, which was injected into the rat's abdominal cavity once a day, 10 mL / kg for a period of 20 days of basic sensitization, and 1 mg / mL ovalbumin saline solution was dripped into the nasal cavity once a day, 10 mL / time. Within 1 hour after the last sensitization, the quantitative superposition scoring method was used for scoring, and the scoring criteria for mouse behavioral symptoms are shown in Table 1. The average total score of the modeling group rats exceeded 5, indicating that the AR rat modeling was successful, and all the rats participating in the modeling experiment were successfully modeled.

[0119] Table 1

[0120] Grading Sneeze(pcs / 30min) Runny nose Scratching nose 1 point 1-3 Anterior nares only Mild nose scratching 2 points 4-10 Beyond the anterior nostril Frequent nose scratching 3 points >11 Runny nose Continuous nose scratching

[0121] Grouped administration: The rats were divided into a healthy group, a model group, Example 1-7 groups, and Comparative Example 1-10 groups, with 10 rats in each group. The Example 1-7 groups and Comparative Example 1-10 groups were sprayed with 10 mL / kg of the Chinese medicine composition for treating allergic rhinitis, and the healthy group and the model group were sprayed with the same amount of normal saline, and the intervention was continued for 30 days.

[0122] 1. Observation of behavioral symptoms. After the experiment, the mice were observed for sneezing, runny nose, scratching nose and other symptoms on the 31st day and scored. The scoring criteria for the behavioral symptoms of mice are shown in Table 1. The results are shown in Table 2.

[0123] Table 2

[0124]

[0125]

[0126] It can be seen from the above table that the Chinese medicinal compositions for treating allergic rhinitis prepared in Examples 1-3 of the present invention can significantly improve the symptoms of rats with rhinitis.

[0127] 2. ELISA was used to detect serum tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), immunoglobulin E (IgE), and recombinant human vascular endothelial growth factor (HIS). Blood samples were collected from rat tail veins, and plasma was separated by centrifugation. The levels of IL-4, HIS, IgE, and TNF-α in plasma were detected by ELISA. The operation was carried out strictly in accordance with the instructions of the kit.

[0128] 3. Detection of eosinophils (EOS) in nasal mucosa. After HE staining, select 5 fields of view (area 675.2 mm × 498 mm) under a microscope and count EOS based on the presence of lobed nuclei and eosinophilic granules in the cytoplasm. 5 EOS / pathological section, 1 EOS counted.

[0129] The results are shown in Table 3.

[0130] Table 3

[0131]

[0132]

[0133]

[0134] Note: * compared with the healthy group, P < 0.05; # compared with the model group, P < 0.05.

[0135] The production of AR is caused by the contact of allergens with the body, and is a type I allergic reaction caused by the massive release of HIS and related mediators such as plasmin triggered by IgE, which triggers sneezing, runny nose and other rhinitis reactions by combining with a variety of immune factors. As can be seen from the above table, the Chinese medicine composition for treating allergic rhinitis prepared in Examples 1-3 of the present invention can significantly reduce the levels of IL-4, TNF-α, IgE, HIS in serum and the number of EOS cells in nasal mucosal tissue.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a Chinese medicine composition for treating allergic rhinitis, characterized in that: The specific steps include: S1. Isolation and extraction of γδT cells: 1-2wt% sodium heparin is added to the peripheral blood for anticoagulation, and mononuclear cells are separated by density gradient centrifugation with lymphocyte separation fluid, and culture medium is added, wherein the mass ratio of mononuclear cells to culture medium is 10-15:100, and proliferation is induced and cultured for 2-4 days at 36-38°C and 4-6% CO2, and the cell pellet is obtained by centrifugation and washed with physiological saline to obtain γδT cells; The culture medium is a 1640 culture medium containing 2-4 mmol / L glutamine and 10-15 mg / L zoledronic acid; S2. Preparation of Ag-graphene oxide particles adsorbing γδT cells: add 2-4 parts by weight of silver nitrate and 3-5 parts by weight of glucose to 100 parts by weight of graphene oxide aqueous dispersion, heat to 85-95°C, stir to react for 0.5-1h, dialyze, freeze-dry to obtain Ag-graphene oxide powder, add 5-7 parts by weight of Ag-graphene oxide powder to 100 parts by weight of physiological saline, add 3-5 parts by weight of γδT cells obtained in step S1, stir to mix evenly, freeze-dry to obtain Ag-graphene oxide particles adsorbing γδT cells; S3. Preparation of Chinese medicine fermentation products: 3-5 parts by weight of Xanthium sibiricum, 2-4 parts by weight of Astragalus, 2-4 parts by weight of Poria, 1-2 parts by weight of Magnolia, 0.5-1 parts by weight of mint were washed, dried, crushed, added to 100 parts by weight of water, sterilized, inoculated with Lactobacillus plantarum and Bifidobacterium infantis seed solution, the inoculation amounts were 1-2v / v% and 2-3v / v%, respectively, the seed solution containing bacteria was 10 8 -10 9 cfu / mL, under anaerobic conditions, 100-150r / min, 36-38°C, fermentation culture for 36-48h, filtration, solid washing, concentration to obtain concentrated bacterial solution, lyophilization of the filtrate, and preparation of Chinese medicine fermentation products; S4. Preparation of eutectic powder: 1-2 parts by weight of berberine was dissolved in 50 parts by weight of chloroform, 3-5 parts by weight of quercetin was dissolved in 50 parts by weight of dimethyl sulfoxide, 2-4 parts by weight of matrine was dissolved in 50 parts by weight of ethanol, the three solutions were mixed uniformly, the solvent was evaporated at room temperature for 2-4d, filtered, washed, ball milled for 1-2h, and dried to obtain eutectic powder; S5. Preparation of a Chinese medicine composition for treating allergic rhinitis: 3-5 parts by weight of the Ag-graphene oxide particles for adsorbing γδT cells prepared in step S2, 15-20 parts by weight of the Chinese medicine fermentation product prepared in step S3, 10-20 parts by weight of the concentrated bacterial solution prepared in step S3, and 0.5-1 parts by weight of the eutectic powder prepared in step S4 are mixed evenly, 100 parts by weight of water, 12-15 parts by weight of carboxymethyl chitosan, 2-3 parts by weight of poloxamer 407 and 1-2 parts by weight of poloxamer 188 are added, mixed evenly, and expanded at 2-4°C for 18-24 hours to prepare a Chinese medicine composition for treating allergic rhinitis.

2. A Chinese medicine composition for treating allergic rhinitis prepared by the preparation method as claimed in claim 1.

3. Use of the Chinese medicine composition for treating allergic rhinitis as claimed in claim 2 in the preparation of medicines for alleviating, improving and treating allergic rhinitis.

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