Traditional Chinese medicine composition for preventing and treating allergic rhinitis and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicines, and relates to a traditional Chinese medicine composition for preventing and treating allergic rhinitis and application thereof. The traditional Chinese medicine composition is prepared by processing honey-fried licorice root, ginseng and ginger.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine and relates to a traditional Chinese medicine composition for preventing and treating allergic rhinitis and application thereof. Background Art

[0002] Allergic rhinitis (AR) is a widespread disease worldwide. It is an inflammatory disease of the nasal mucosa caused by allergen stimulation and IgE-mediated release of histamine as the main inflammatory mediator. The main clinical symptoms are nasal itching, paroxysmal sneezing, runny nose and nasal congestion. 60%-70% of patients also have eye symptoms, such as inflammatory symptoms such as itching and tearing, and are prone to recurrence.

[0003] Allergic rhinitis is called nasal congestion in traditional Chinese medicine. Traditional Chinese medicine believes that the internal cause of allergic rhinitis is the deficiency of internal organs (lungs, spleen and kidneys), and the external cause is the invasion of wind-cold evil or foreign air. In clinical allergic rhinitis, it is very easy for the evil to enter the interior without being resolved on the surface, and the constitution of Shaoyang patients is prone to cold and heat, and to deficiency and excess. When they are sick, they often have a mixture of cold and heat, deficiency and excess, and a mixed picture of cold, heat, deficiency and excess. In treatment, simply resolving the exterior is easy to cause exterior deficiency, and strengthening the foundation is not conducive to eliminating evil. Neither tonic nor purgative should be used indiscriminately. Resolving the exterior or clearing the interior cannot accurately achieve the purpose of treatment. Therefore, the "harmony method" is mainly used, and the method of using both cold and heat, eliminating and replenishing, and treating both the exterior and the interior is adopted to achieve the purpose of eliminating cold without generating heat, clearing heat without generating cold, replenishing without hindering evil, and eliminating without hurting the body.

[0004] At present, the drugs used in clinical practice can only control the symptoms of allergic rhinitis, but cannot achieve the purpose of radical cure, and there are many adverse reactions. Traditional Chinese medicine focuses on the overall concept and syndrome differentiation and treatment, which can treat the symptoms while also treating the root cause. This project uses modern scientific theories to explain and demonstrate ancient Chinese wisdom, and based on the combined scores of the active ingredients of each medicine in the classic prescription and the disease targets of allergic rhinitis, screens key components for combination, and provides new ideas for the treatment of allergic rhinitis with traditional Chinese medicine. Summary of the invention

[0005] The object of the present invention is to provide a Chinese medicine composition for preventing and / or treating allergic rhinitis.

[0006] At present, the side effects of drugs for treating allergic rhinitis are large. Applying the theory of traditional Chinese medicine to analyze the cause of the disease, it is found that patients with allergic rhinitis have mixed cold and heat, deficiency and excess, and a mixed phenomenon of cold and heat, deficiency and excess. Therefore, the "harmony method" is mainly used, and the methods of using both cold and heat, eliminating and replenishing, and treating both the exterior and the interior are adopted to achieve the purpose of eliminating cold without generating heat, clearing heat without generating cold, replenishing without hindering evil, and eliminating without hurting the body. The traditional Chinese medicine compound Xiao Chaihu Decoction is the "ancestor of the harmony prescription". The traditional Chinese medicine compound Xiao Chaihu Decoction is a classic prescription with a long history of use and its safety has been proven. This project screened out three medicines, namely, licorice, ginseng and ginger, which may play a key role in the treatment of allergic rhinitis based on the combination score of the active ingredients of each medicine in the traditional Chinese medicine compound Xiao Chaihu Decoction and the disease targets of allergic rhinitis. The in vivo experiment further confirmed that the combination of licorice, ginseng and ginger has a significant therapeutic effect on allergic rhinitis.

[0007] The Chinese medicine composition of the present invention is processed from the following ingredients in parts by weight:

[0008] 4.5-18 weight portions of licorice root

[0009] 4.5-18 parts by weight of ginseng

[0010] 4.5-18 parts by weight of ginger

[0011] Preferably, the Chinese medicine composition of the present invention is processed from the following ingredients in parts by weight:

[0012] 9 parts by weight of licorice root

[0013] Ginseng 9 parts by weight

[0014] 9 parts by weight of ginger

[0015] The unit of weight parts may be g, kg, etc.

[0016] Another object of the present invention is to provide a method for preparing the pharmaceutical composition.

[0017] (1) Soaking in cold water: Place the herbs in an appropriate amount of cold water, making sure that the water level is enough to cover the herbs by 2-5 cm to prevent them from drying out or burning. The soaking time is 20-60 minutes.

[0018] (2) Boil over high heat: Put the soaked herbs into a casserole, add 150-600 mL of water, and boil over high heat;

[0019] (3) Boil over low heat: When the liquid medicine boils, turn to low heat and continue boiling. Start timing at this time. Boil for 15-60 seconds.

[0020] min;

[0021] (4) Filtering the liquid medicine: After boiling for 30 minutes, filter the liquid medicine through a sieve and pour it into a beaker. Put the residue back into the casserole, add 150-600 mL of water, boil again, and then boil over low heat for 15-60 minutes.

[0022] (5) Mixing the medicinal liquid: Filter out the medicinal liquid from the second boiling, combine it with the medicinal liquid from the first boiling, and pour it into a casserole;

[0023] (6) Evaporation and concentration: The combined medicinal solution is evaporated and concentrated in a casserole to obtain the product.

[0024] Preferably, the preparation method of the present invention comprises the following steps:

[0025] (1) Soaking in cold water: Place the herbs in an appropriate amount of cold water, making sure that the water level is enough to cover the herbs by 2-5 cm to prevent them from drying out or burning. The soaking time is 40 minutes.

[0026] (2) Boil over high heat: Place the soaked herbs in a casserole, add 300 mL of water, and boil over high heat.

[0027] (3) Boil over low heat: After the medicinal liquid boils, turn to low heat and continue boiling. Start timing at this time and boil for 30 minutes. During the boiling process, you need to stir continuously with a glass rod to ensure that the medicinal materials are fully cooked.

[0028] (4) Filter the liquid medicine: After boiling for 30 minutes, filter the liquid medicine through a sieve and pour it into a beaker. Put the residue back into the casserole, add 300 mL of water, boil again, then simmer for 30 minutes on low heat. During this process, you also need to stir constantly.

[0029] (5) Mixing the medicinal liquid: Filter out the medicinal liquid from the second boiling, combine it with the first boiling liquid, and pour it into a casserole.

[0030] (6) Evaporation and concentration: The combined medicinal solution is evaporated and concentrated in a casserole to obtain the product.

[0031] Another object of the present invention is to provide use of the pharmaceutical composition in preparing a medicament for preventing and treating allergic rhinitis.

[0032] The present invention proposes that the Chinese medicine compound of licorice, ginseng and ginger can significantly inhibit the number of sneezing and scratching of the nose in mice with allergic rhinitis, and significantly inhibit the levels of IgE and histamine in the serum of mice with allergic rhinitis. The Chinese medicine compound of licorice, ginseng and ginger can significantly reduce the thickening of the nasal mucosa caused by allergic rhinitis, increase the expression of the tight junction protein Occludin and the cytoplasmic tight adhesion protein ZO-1 in the nasal mucosa, Occludin and ZO-1 play an important role in maintaining the integrity of nasal epithelial cells, regulating permeability and participating in cell signal transduction. Restoring the normal levels of these two proteins is crucial to protecting the nasal mucosa from allergens and pathogens, and helps repair the epithelial barrier and alleviate symptoms. The Chinese medicine compound of licorice, ginseng and ginger can significantly reduce the number of goblet cells and mast cells on the nasal mucosa, and inhibit the levels of inflammatory factors IL-4, IL-5 and IL-13 in the serum. The Chinese herbal compound of licorice, ginseng and ginger had no significant effect on the tissue structure of the liver and kidney. The expression levels of serum alanine aminotransferase ALT and urea nitrogen BUN also confirmed that the Chinese herbal compound of licorice, ginseng and ginger had no significant effect on liver function and kidney function.

[0033] The composition of the present invention is in the form of a pharmaceutical preparation, and the pharmaceutical preparation may further include pharmaceutical excipients as required.

[0034] The pharmaceutical preparation of the present invention can be prepared into any pharmaceutically acceptable dosage form, including oral and external dosage forms, selected from: tablets, capsules, lozenges, granules, pills, powders, ointments, pills, suspensions, solutions, injections, suppositories, ointments, sprays, drops (nasal drops), pills or patches.

[0035] The pharmaceutical composition of the present invention, its oral administration preparation may contain common excipients, such as binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents and wetting agents, and the tablets may be coated if necessary.

[0036] Suitable fillers include cellulose, mannitol, lactose and other similar fillers. Suitable disintegrants include starch, polyvinyl pyrrolidone and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.

[0037] The pharmaceutical composition of the present invention can be prepared into a solid oral composition by conventional methods such as mixing, filling, tableting, etc. Repeated mixing can distribute the active substance throughout those compositions using a large amount of fillers.

[0038] Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be a dry product that can be reconstituted with water or other suitable carriers before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, for example lecithin, sorbitan monooleate or gum arabic; non-aqueous carriers (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerol esters, propylene glycol or ethanol; preservatives, for example methylparaben or propylparaben or sorbic acid, and, if desired, conventional flavoring or coloring agents.

[0039] For injection, a liquid unit dosage form is prepared containing the active substance of the invention and a sterile carrier. Depending on the carrier and concentration, the compound can be suspended or dissolved. Solutions are usually prepared by dissolving the active substance in a carrier, filtering and sterilizing it before filling it into a suitable vial or ampoule, and then sealing it. Excipients such as a local anesthetic, preservatives and buffers can also be dissolved in this carrier. In order to improve its stability, the composition can be frozen after filling into the vial and the water removed under vacuum.

[0040] The pharmaceutical composition of the present invention can be selectively added with a suitable pharmaceutically acceptable carrier when it is prepared into a medicament. The pharmaceutically acceptable carrier is selected from the group consisting of mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, soil temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The drug formulation and its therapeutic effect of the present invention are obtained through a large number of experimental screenings:

[0043] 1. Screening of key components in Xiao Chaihu Decoction for the treatment of allergic rhinitis

[0044] To screen out the key components of Xiao Chai Hu Tang for the treatment of allergic rhinitis, a network pharmacology method was used for systematic analysis. The main components of each medicinal material of Xiao Chai Hu Tang were retrieved using the TCMSP database (https: / / old.tcmsp-e.com). Potentially active ingredients were screened out according to the screening criteria of bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The Swiss Target Prediction database (http: / / swisstargetprediction.ch / ) was used to predict the potential therapeutic targets of these active ingredients, and the potential therapeutic targets of the active ingredients were further evaluated based on pharmacokinetics (GI absorption: high) and drug-likeness (Lipinski's five rules and Ghose's law).

[0045] In the Genecards database (http: / / www.genecards.org), OMIM database (http: / / omim.org / ) and DisGeNET database (http: / / www.disgenet.org / web / DisGeNET / ), the key words "allergic rhinitis" were used to screen out genes related to allergic rhinitis. The Venny 2.1 tool was used to construct the intersection of potential therapeutic targets of Xiao Chaihu Decoction and genes related to allergic rhinitis, and to determine the key genes for Xiao Chaihu Decoction in the treatment of allergic rhinitis.

[0046] Cytoscape was used to construct a drug component-target network diagram based on the intersection genes corresponding to the active ingredients in Xiao Chaihu Decoction. The top ten active ingredients that may have great potential for the treatment of allergic rhinitis were screened out according to the degree value. The results showed that these active ingredients were mainly concentrated in licorice, ginseng and ginger. Therefore, the present invention combined licorice, ginseng and ginger as a traditional Chinese medicine composition for subsequent animal experiments to verify its efficacy.

[0047] 2. Construction of allergic rhinitis mouse model and drug administration

[0048] The sensitizer chicken ovalbumin (OVA) and the adjuvant aluminum hydroxide were used to establish an allergic rhinitis mouse model, and the ELISA kit was used to detect the IgE level in the serum and the behavioral changes to verify whether the allergic rhinitis mouse model was successfully established.

[0049] Establishment of a mouse model of allergic rhinitis

[0050] Eight-week-old male BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. On days 1, 8, and 15, 200 μl of PBS containing 25 μg of sensitizer chicken egg albumin OVA and 2 mg of adjuvant aluminum hydroxide were intraperitoneally injected. On days 22-35, 20 μl of PBS containing 25 mg / mL OVA was infused intranasally. After the last nasal challenge on day 35, the mice were immediately observed and scored for the number of nose scratching, sneezing, and runny nose within 20 minutes. Sneezing: 1 point for 1-3 times; 2 points for 4-10 times; 3 points for more than 11 times. Scratching the nose: 1 point for gently rubbing the nose a few times; 2 points for frequent nose scratching; 3 points for continuous nose scratching with both forelimbs. Runny nose: 1 point for flowing to the anterior nares, 2 points for exceeding the anterior nares, and 3 points for runny nose all over the face. The above three symptom scores were added together, and a total score greater than 5 points indicated a successful modeling. The model establishment process is as follows: Figure 1 shown.

[0051] Grouping and dosing

[0052] The experimental groups were: normal control group, allergic rhinitis mouse group, Xiao Chaihu Decoction treatment group, Chinese medicine compound (Glycyrrhiza uralensis, ginseng, ginger) treatment group, and loratadine positive control group. Each group had 20 mice, divided into 2 cages, 10 mice in each cage. The configuration of Xiao Chaihu Decoction: Bupleurum 15g, Scutellaria baicalensis 9g, Pinellia ternata 9g, Glycyrrhiza uralensis 9g, Glycyrrhiza uralensis 9g, Ginseng slices 9g, Ginger 9g, Jujube 6g. To ensure the safety of medication, the dosage of the Chinese medicine compound administration group was still configured according to the dosage of Xiao Chaihu Decoction, and the dosage of the Chinese medicine compound was: Glycyrrhiza uralensis 9g, Ginseng slices 9g, Ginger 9g. The configuration method was: (1) Cold water soaking: Put the medicinal materials into an appropriate amount of cold water, and make sure that the water volume is enough to cover the medicinal materials by 2-5cm to prevent the medicinal materials from being boiled dry or burned. The soaking time was 40min. (2) Boil over high heat: Put the soaked herbs into a casserole, add 300 mL of water, and boil over high heat. (3) Boil over low heat: After the medicinal liquid boils, turn to low heat and continue boiling. Start timing at this time and boil for 30 minutes. During the decoction process, you need to stir continuously with a glass rod to ensure that the herbs are fully cooked. (4) Filter the medicinal liquid: After boiling for 30 minutes, use a sieve to filter out the medicinal liquid and pour it into a beaker. Put the filtrate back into the casserole, add 300 mL of water, boil again, and then turn to low heat and boil for 30 minutes. During this process, you also need to stir continuously. (5) Mix the medicinal liquid: Filter the medicinal liquid from the second boiling, combine it with the medicinal liquid from the first boiling, and pour it into the casserole. (6) Evaporation and concentration: Evaporate and concentrate the combined medicinal liquid in the casserole. Then, let the medicinal liquid cool and divide it into 15 mL centrifuge tubes. Finally, store the centrifuge tubes in a -20℃ refrigerator. The drug was administered by gavage on days 1-3, 8-10, 15-17, and 22-35. The dosage was calculated based on the ratio of human to mouse body surface area, with a conversion coefficient of 9.1 for adults and mice. Based on a 70kg adult taking one dose of medicine per day, the single dosage of mice in the Xiao Chaihu Decoction group was 8.58 g / kg, and the single dosage of mice in the Chinese medicine compound group was 3.51 g / kg. After conversion, the dosage of the decoction was 0.2 mL. The dosage of loratadine in the positive control group was 1.5 mg / kg, and the frequency and time of administration were the same as those in the Chinese medicine compound treatment group.

[0053] In order to verify whether the effect of the Chinese herbal compound on inhibiting allergic rhinitis is dose-dependent, high-, medium-, and low-dose treatment groups of the Chinese herbal compound were set up, with the dosages of 7.02 g / kg, 3.51 g / kg, and 1.76 g / kg, respectively. The high-dose group was configured with 18 g of licorice root, 18 g of ginseng slices, and 18 g of ginger. The medium-dose group (consistent with the dosage of Xiao Chaihu Decoction) was configured with 9 g of licorice root, 9 g of ginseng slices, and 9 g of ginger. The low-dose group was configured with 4.5 g of licorice root, 4.5 g of ginseng slices, and 4.5 g of ginger. The configuration method is as follows: (1) Soak in cold water: Put the medicinal materials into an appropriate amount of cold water, making sure that the water volume is enough to cover the medicinal materials by 2-5 cm to prevent the medicinal materials from being boiled or burned. The soaking time is 40 min. (2) Boil over high heat: Put the soaked medicinal materials into a casserole, add 300 mL of water, and boil over high heat. (3) Boil on low heat: After the medicinal liquid boils, turn to low heat and continue boiling. Start timing at this time and boil for 30 minutes. During the decoction process, it is necessary to stir continuously with a glass rod to ensure that the medicinal materials are fully cooked. (4) Filter the medicinal liquid: After boiling for 30 minutes, use a sieve to filter out the medicinal liquid and pour it into a beaker. Put the filter residue back into the casserole, add 300mL of water, boil again, and then boil on low heat for 30 minutes. During this process, it is also necessary to stir constantly. (5) Mix the medicinal liquid: Filter the medicinal liquid from the second boil, combine it with the first medicinal liquid, and pour it into the casserole. (6) Evaporation and concentration: Evaporate and concentrate the combined medicinal liquid in a casserole. Then, let the medicinal liquid cool and divide it into 15mL centrifuge tubes. Finally, store the centrifuge tubes in a -20℃ refrigerator. The frequency and time of administration are similar to Figure 1 The timeline in A is consistent.

[0054] ELISA kit to detect mouse serum IgE and histamine levels

[0055] ELISA kits were used to detect the levels of IgE and histamine in mouse serum to evaluate whether the establishment of the mouse model of allergic rhinitis was successful and the severity of the allergic reaction. After the mice were anesthetized, blood was collected from the eyeballs and collected into 1.5mL EP tubes. The blood was allowed to stand at room temperature for 1h until it naturally coagulated, centrifuged at 4000rpm for 10min, and the serum was collected into new EP tubes. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20min, and the standard wells and sample wells were set up. 50μL of standards of different concentrations were added to each standard well. 10μL of the sample to be tested was first added to the sample well, and then 40μL of the sample diluent was added; no addition was made to the blank well. In addition to the blank well, 100μL of the detection antibody labeled with horseradish peroxidase was added to each well of the standard well and the sample well, and the reaction wells were sealed with a sealing film, and incubated in a 37℃ constant temperature incubator for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times. Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value and then multiply it by 5 to obtain the actual concentration of IgE and histamine.

[0056] 3. HE staining to evaluate pathological changes in mouse nasal tissue

[0057] HE staining was used to observe the changes in nasal tissue morphology and nasal mucosal thickness of mice, and to evaluate the effect of the Chinese medicine compound on the nasal mucosa of mice with allergic rhinitis. The tissue sections were first dewaxed, and the sections were immersed in xylene I and II for 10 minutes each; then hydrated, and the sections were immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 1 minute each, and then rinsed with double distilled water for 2 minutes; then stained, the sections were immersed in hematoxylin stain for 1 minute, gently rinsed with tap water for 2 minutes, rinsed with running water for 1 minute, and then separated with 1% hydrochloric acid alcohol for 2 seconds, rinsed with running water for 5 minutes, and finally stained with eosin for 10 seconds and rinsed with running water for 1 minute; then dehydrated and transparentized, the sections were immersed in 75% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 1 minute each, and then immersed in xylene I and II for 2 minutes each; finally, the sections were sealed with neutral resin, observed under a microscope and images were collected.

[0058] 4. Immunofluorescence staining to assess the integrity of mouse nasal epithelium

[0059] Immunofluorescence staining was used to detect the expression levels of tight junction protein Occludin and cytoplasmic tight adhesion protein ZO-1 in mouse nasal mucosa and evaluate the integrity of mouse nasal epithelium. First, paraffin sections were baked at 60℃ for 1.5h to promote tissue adhesion; dewaxed and hydrated with xylene and gradient ethanol (100%, 95%, 85%) in sequence, each step for 5min, and washed with distilled water; antigen retrieval was performed with sodium citrate, and the sections were naturally cooled after heating in a high-temperature microwave oven, and washed in PBS for 3 times, each time for 5min; blocked with 3% BSA for 30min to prevent nonspecific binding; primary antibody was added and incubated at 4℃ overnight; washed with PBS 3 times, each time for 5min; secondary antibody was added and incubated at room temperature in the dark for 1h; washed with PBS 3 times, each time for 5min; nuclear staining reagent DAPI was added and incubated in the dark for 10min; washed with PBS 3 times, each time for 5min; anti-fluorescence quenching sealing agent was used to seal the sections, and the images were observed and collected using a fluorescence microscope.

[0060] 5. PAS staining to evaluate the level of mouse nasal mucosal goblet cells

[0061] PAS staining was used to detect the number of goblet cells in the nasal mucosa of mice and to evaluate the abnormal mucus secretion function of mice. After the sections were dewaxed and hydrated, the periodic acid solution was taken out and balanced to room temperature, and 100 μl of periodic acid solution was added to each sample, and the reaction was carried out in the dark for 5 minutes; the periodic acid solution was removed, and the sections were immersed in distilled water and washed on a shaker for 5 minutes; 100 μL Schiff reagent was added, and the sections were placed in a wet box and stained in a 37°C incubator in the dark for 5 minutes; the staining solution was removed and the sections were immersed in distilled water and washed on a shaker for 5 minutes; the sections were treated with gradient ethanol and xylene, and after proper drying, the sections were sealed with neutral gum; and the images were observed and collected under a microscope.

[0062] 6. Giemsa staining to assess the level of mast cells in mouse nasal mucosa

[0063] Giemsa staining was used to detect the number of mast cells in the mouse nasal mucosa and evaluate the level of nasal mucosal inflammation in mice. After dewaxing and hydration, the sections were added with 200 μl of Giemsa working solution and stained for 1 min; rinsed thoroughly with distilled water from one side; treated with gradient ethanol and xylene, dried and sealed with neutral gum, and observed and collected images under a microscope.

[0064] 7. Calculation of spleen index of mice with allergic rhinitis

[0065] During the culture period of the allergic rhinitis mouse model, the weight of the mice was recorded every 7 days. After the model was established and the administration was completed, the mice were killed and the spleen was removed. The surface moisture of the spleen was absorbed with filter paper and then weighed. The spleen index was calculated using the final body weight and spleen weight: spleen index = spleen weight (g) / body weight (g) to evaluate the immune response level of the mice.

[0066] 8. ELISA kit to detect the levels of IL-4, IL-5 and IL-13 in mouse serum

[0067] ELISA kits were used to detect the levels of IL-4, IL-5 and IL-13 in mouse serum and to evaluate the level of nasal mucosal inflammation in mice. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20 minutes, and standard wells and sample wells were set. 50 μL of standard wells with different concentrations were added. 10 μL of the sample to be tested was first added to the sample well, and then 40 μL of sample diluent was added; no addition was made to the blank well. Except for the blank well, 100 μL of detection antibody labeled with horseradish peroxidase was added to each well of the standard well and sample well, and the reaction wells were sealed with a sealing film and incubated in a 37°C incubator for 60 minutes. The liquid was discarded, patted dry on absorbent paper, and each well was filled with washing solution, left to stand for 1 minute, the washing solution was shaken off, and patted dry on absorbent paper. The washing plate was washed 5 times. 50 μL of substrate A and B were added to each well, and incubated at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value and then multiply it by 5 to obtain the actual concentration of IL-4, IL-5 and IL-13.

[0068] 9. HE staining to evaluate the effect of Chinese herbal medicine on liver and kidney tissues of mice

[0069] HE staining was used to evaluate the toxicity of the Chinese medicine compound on the kidney and liver of mice. The tissue sections were first dewaxed and immersed in xylene I and II for 10 min each; then hydrated and immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 1 min each, and then rinsed with double distilled water for 2 min; then stained and immersed in hematoxylin stain for 1 min, gently rinsed with tap water for 2 min, rinsed with running water for 1 min, then separated with 1% hydrochloric acid alcohol for 2 s, rinsed with running water for 5 min, and finally stained with eosin for 10 s and rinsed with running water for 1 min; then dehydrated and transparentized, immersed in 75% ethanol, 80% ethanol, 95% ethanol, and anhydrous ethanol for 1 min each, and then immersed in xylene I and II for 2 min each; finally, the sections were sealed with neutral resin, observed under a microscope, and images were collected.

[0070] 10. ELISA kit to detect the liver and kidney function levels of mice

[0071] ELISA kits were used to detect the levels of serum alanine aminotransferase ALT and urea nitrogen BUN in mice to evaluate the toxicity of the Chinese medicine compound on the liver and kidney of mice. ALT: 20 μL of matrix solution was preheated to 37°C; 20 μL of matrix solution and 5 μL of the sample to be tested were added to the assay wells, and 20 μL of matrix solution was added to the control wells; the microplate was gently shaken to mix, and the reaction was carried out at 37°C for 30 min; 20 μL of 2,4-dinitrophenylhydrazine solution was added to the assay wells, and 5 μL of 2,4-dinitrophenylhydrazine solution was added to the control wells; the microplate was gently shaken to mix, and the reaction was carried out at 37°C for 20 min; 200 μL of 0.4 mol / L sodium hydroxide solution was added to each of the assay wells and the control wells; the microplate was gently shaken to mix, and the mixture was placed at room temperature for 15 min; the wavelength was set to 505 nm, the OD value of each well was measured, and the net OD value was obtained by subtracting the OD value of the control well from the OD value of the assay well; the standard curve was substituted to calculate the ALT activity value. BUN: Add 0.02mL of distilled water to the blank tube, add 0.02mL of 10mmol / L BUN standard application solution to the standard tube, and add 0.02mL of the sample to be tested to the measurement tube; add 0.25mL of buffered enzyme solution to each tube; mix well and incubate in a 37℃ constant temperature box for 10min; add 1mL of color developer and 1mL of alkaline sodium hypochlorite to each tube; mix well and incubate in a 37℃ constant temperature box again for 10min; set the wavelength to 640nm, the light path to 1cm, adjust to zero with distilled water, measure the absorbance OD value of each tube, and substitute into the formula to obtain the urea nitrogen concentration: urea nitrogen concentration (mmol / L) = (A 测定 -A 空白 ) / (A 标准 -A 空白 )×C 标准。

[0072] 11. Statistical analysis

[0073] The experimental data were expressed as mean ± standard deviation and statistically analyzed using SPSS17.0. The t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison between multiple groups. When there were differences in ANOVA analysis, the Bonferroni-corrected t-test or Dunnett t-test was used to evaluate whether there were differences between the groups. P < 0.05 was considered to be statistically significant.

[0074] Explanation of some English abbreviations or terms in the manual:

[0075] IgE: Immunoglobulin E

[0076] XCHT: Xiao Chaihu Decoction

[0077] AR: Allergic rhinitis

[0078] Zhi: Roasted Licorice

[0079] Ren: Ginseng

[0080] Sheng: ginger

[0081] Jiang:Ginger Pinellia

[0082] Occludin: tight junction protein

[0083] ZO-1: cytoplasmic tight adhesion protein

[0084] Degree: degree

[0085] Betweenness

[0086] Closeness: Closeness to centrality

[0087] OVA: ovalbumin

[0088] ALT: Alanine transaminase

[0089] BUN: urea nitrogen

[0090] IL-4: interleukin 4

[0091] IL-5: interleukin 5

[0092] IL-13: interleukin 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 .Screening of key components in Xiao Chaihu DecoctionA. The intersection of potential therapeutic targets of Xiao Chaihu Decoction and related genes for allergic rhinitisB. Drug component-target network diagramC. Degree value ranking of active ingredientsXCHT: Xiao Chaihu DecoctionAR: Allergic rhinitisZhi: Zhi GancaoRen: GinsengSheng: Sheng JiangJiang: Pinellia ternata. Figure 2 .Establishment and drug administration of allergic rhinitis mouse modelA. Timeline of establishment of allergic rhinitis mouse model and drug administration timeB. Serum IgE levelC. Serum histamine levelD. Number of sneezesE. Number of nose scratchingCTL: normal control group miceAR: allergic rhinitis miceXCHT: Xiao Chaihu Decoction treated miceHTCM: high-dose Chinese herbal compound roasted licorice, ginseng and ginger treated miceMTCM: medium-dose Chinese herbal compound roasted licorice, ginseng and ginger treated mice (consistent with Xiao Chaihu Decoction dose)LTCM: low-dose Chinese herbal compound roasted licorice, ginseng and ginger treated miceLor: loratadine positive control group mice*P<0.05, **P<0.01, ****P<0.0001.

[0094] Figure 3.HE staining to evaluate the pathological changes of nasal tissue in miceA. Nasal tissue morphologyB. Nasal mucosal thicknessCTL: normal control group miceAR: allergic rhinitis miceTCM: Chinese medicine compound licorice, ginseng and ginger treatment group miceLor: loratadine positive control group mice****P<0.0001.

[0095] Figure 4 Immunofluorescence staining to evaluate the integrity of mouse nasal epithelium Expression of nasal mucosal tight junction protein Occludin and cytoplasmic tight adhesion protein ZO-1 in mouse nasal mucosal tissue CTL: normal control mice AR: allergic rhinitis mice TCM: Chinese medicine compound licorice, ginseng and ginger treatment group mice Lor: loratadine positive control group mice

[0096] Figure 5 .PAS staining to detect goblet cells in the nasal mucosa of mice CTL: Normal control mice AR: Allergic rhinitis mice TCM: Chinese medicine compound licorice, ginseng and ginger treatment group mice Lor: Loratadine positive control group mice

[0097] Figure 6 .Giemsa staining to detect mast cells in mouse nasal mucosa CTL: Normal control mice AR: Allergic rhinitis mice TCM: Chinese medicine compound licorice, ginseng and ginger treatment group mice Lor: Loratadine positive control group mice

[0098] Figure 7 .Spleen index of mice with allergic rhinitisA. Mouse spleenB. Spleen indexCTL: mice in normal control groupAR: mice with allergic rhinitisTCM: mice in the treatment group with the Chinese medicine compound licorice, ginseng and gingerLor: mice in the loratadine positive control group****P<0.0001.

[0099] Figure 8 .ELISA kit was used to detect the levels of IL-4, IL-5 and IL-13 in mouse serumA. The level of IL-4 in serumB. The level of IL-5 in serumC. The level of IL-13 in serumCTL: mice in normal control groupAR: mice in allergic rhinitisTCM: mice in the treatment group with the Chinese medicine compound licorice, ginseng and gingerLor: mice in the loratadine positive control group**P<0.01, ***P<0.001, ****P<0.0001.

[0100] Fig. 9 HE staining to evaluate the effect of the Chinese herbal compound on the liver and kidney tissues of mice A. HE staining of liver tissue B. HE staining of kidney tissue CTL: mice in the normal control group AR: mice with allergic rhinitis TCM: mice in the treatment group with the Chinese herbal compound licorice, ginseng and ginger

[0101] Fig.10.Animal behavior experiment to evaluate the effects of other components of Xiao Chaihu Decoction on mice with allergic rhinitisA. Serum IgE levelB. Serum histamine levelC. Number of sneezesD. Number of nose scratchingCTL: Normal control group miceAR: Allergic rhinitis miceXCHTO: Xiao Chaihu Decoction other components treatment group miceLor: Loratadine positive control group mice****P<0.0001.

[0102] Fig.11 .Experimental technology roadmap. DETAILED DESCRIPTION

[0103] The present invention is further illustrated by the following examples.

[0104] The product and preparation method of the present invention are further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0105] Example 1. Screening of key components in Xiao Chaihu Decoction for treating allergic rhinitis

[0106] To screen out the key components of Xiao Chai Hu Tang for the treatment of allergic rhinitis, a network pharmacology method was used for systematic analysis. The main components of each medicinal material of Xiao Chai Hu Tang were retrieved using the TCMSP database (https: / / old.tcmsp-e.com). Potentially active ingredients were screened out according to the screening criteria of bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. The Swiss Target Prediction database (http: / / swisstargetprediction.ch / ) was used to predict the potential therapeutic targets of these active ingredients, and the potential therapeutic targets of the active ingredients were further evaluated based on pharmacokinetics (GI absorption: high) and drug-likeness (Lipinski's five rules and Ghose's law).

[0107] In the Genecards database (http: / / www.genecards.org), OMIM database (http: / / omim.org / ) and DisGeNET database (http: / / www.disgenet.org / web / DisGeNET / ), the key words "allergic rhinitis" were used to screen out genes related to allergic rhinitis. The Venny 2.1 tool was used to construct the intersection of potential therapeutic targets of Xiao Chaihu Decoction and genes related to allergic rhinitis, and the key genes for Xiao Chaihu Decoction in treating allergic rhinitis were determined ( Figure 1 A).

[0108] Cytoscape was used to construct a drug component-target network diagram based on the intersection genes corresponding to the active ingredients in Xiao Chaihu Decoction ( Figure 1 B). The top ten active ingredients with great potential for the treatment of allergic rhinitis were screened according to the degree value. The results showed that these active ingredients were mainly concentrated in licorice, ginseng and ginger ( Figure 1 C), therefore, the present invention combines licorice, ginseng and ginger as a Chinese medicine composition to conduct subsequent animal experiments to verify its therapeutic effect.

[0109] Example 2. Establishment of allergic rhinitis mouse model and drug administration

[0110] Eight-week-old male BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., and 200 μl of PBS containing 25 μg of sensitizer chicken ovalbumin OVA and 2 mg of adjuvant aluminum hydroxide were intraperitoneally injected on days 1, 8, and 15. On days 22-35, 20 μl of PBS containing 25 mg / mL OVA was intranasally infused. After the last nasal provocation on day 35, the number of nose scratching and sneezing of the mice within 20 minutes was immediately observed and recorded, and the scores were given. Sneezing: 1-3, 1 point; 4-10, 2 points; more than 11, 3 points. Scratching the nose: 1 point for gently rubbing the nose a few times; 2 points for frequent nose scratching; 3 points for continuous nose scratching with both forelimbs. The above two symptom scores are added together, and a total score greater than 5 points indicates that the model is successfully established. The model establishment process is as follows: Figure 1 As shown in A.

[0111] The experimental groups were: normal control group, allergic rhinitis mouse group, Xiao Chaihu Decoction treatment group, Chinese medicine compound (Glycyrrhiza uralensis, ginseng, ginger) treatment group, and loratadine positive control group. Each group had 20 mice, divided into 2 cages, 10 mice in each cage. The configuration of Xiao Chaihu Decoction: Bupleurum 15g, Scutellaria baicalensis 9g, Pinellia ternata 9g, Glycyrrhiza uralensis 9g, Glycyrrhiza uralensis 9g, Ginseng slices 9g, Ginger 9g, Jujube 6g. To ensure the safety of medication, the dosage of the Chinese medicine compound administration group was still configured according to the dosage of Xiao Chaihu Decoction, and the dosage of the Chinese medicine compound was: Glycyrrhiza uralensis 9g, Ginseng slices 9g, Ginger 9g. The configuration method was: (1) Cold water soaking: Put the medicinal materials into an appropriate amount of cold water, and make sure that the water volume is enough to cover the medicinal materials by 2-5cm to prevent the medicinal materials from being boiled dry or burned. The soaking time was 40min. (2) Boil over high heat: Put the soaked herbs into a casserole, add 300 mL of water, and boil over high heat. (3) Boil over low heat: After the medicinal liquid boils, turn to low heat and continue boiling. Start timing at this time and boil for 30 minutes. During the decoction process, you need to stir continuously with a glass rod to ensure that the herbs are fully cooked. (4) Filter the medicinal liquid: After boiling for 30 minutes, use a sieve to filter out the medicinal liquid and pour it into a beaker. Put the filtrate back into the casserole, add 300 mL of water, boil again, and then turn to low heat and boil for 30 minutes. During this process, you also need to stir continuously. (5) Mix the medicinal liquid: Filter the medicinal liquid from the second boiling, combine it with the medicinal liquid from the first boiling, and pour it into the casserole. (6) Evaporation and concentration: Evaporate and concentrate the combined medicinal liquid in the casserole. Then, let the medicinal liquid cool and divide it into 15 mL centrifuge tubes. Finally, store the centrifuge tubes in a -20℃ refrigerator. The drug was administered by gavage on days 1-3, 8-10, 15-17, and 22-35. The dosage was calculated based on the ratio of human to mouse body surface area, with a conversion coefficient of 9.1 for adults and mice. Based on a 70kg adult taking one dose of medicine per day, the single dosage of mice in the Xiao Chaihu Decoction group was 8.58 g / kg, and the single dosage of mice in the Chinese medicine compound group was 3.51 g / kg. After conversion, the dosage of the decoction was 0.2 mL. The dosage of loratadine in the positive control group was 1.5 mg / kg, and the frequency and time of administration were the same as those in the Chinese medicine compound treatment group.

[0112] In order to verify whether the effect of the Chinese herbal compound on inhibiting allergic rhinitis is dose-dependent, high-, medium-, and low-dose treatment groups of the Chinese herbal compound were set up, with the dosages of 7.02 g / kg, 3.51 g / kg, and 1.76 g / kg, respectively. The high-dose group was configured with 18 g of licorice root, 18 g of ginseng slices, and 18 g of ginger. The medium-dose group (consistent with the dosage of Xiao Chaihu Decoction) was configured with 9 g of licorice root, 9 g of ginseng slices, and 9 g of ginger. The low-dose group was configured with 4.5 g of licorice root, 4.5 g of ginseng slices, and 4.5 g of ginger. The configuration method is as follows: (1) Soak in cold water: Put the medicinal materials into an appropriate amount of cold water, making sure that the water volume is enough to cover the medicinal materials by 2-5 cm to prevent the medicinal materials from being boiled or burned. The soaking time is 40 min. (2) Boil over high heat: Put the soaked medicinal materials into a casserole, add 300 mL of water, and boil over high heat. (3) Boil on low heat: After the medicinal liquid boils, turn to low heat and continue boiling. Start timing at this time and boil for 30 minutes. During the decoction process, it is necessary to stir continuously with a glass rod to ensure that the medicinal materials are fully cooked. (4) Filter the medicinal liquid: After boiling for 30 minutes, use a sieve to filter out the medicinal liquid and pour it into a beaker. Put the filter residue back into the casserole, add 300mL of water, boil again, and then boil on low heat for 30 minutes. During this process, it is also necessary to stir constantly. (5) Mix the medicinal liquid: Filter the medicinal liquid from the second boil, combine it with the first medicinal liquid, and pour it into the casserole. (6) Evaporation and concentration: Evaporate and concentrate the combined medicinal liquid in a casserole. Then, let the medicinal liquid cool and divide it into 15mL centrifuge tubes. Finally, store the centrifuge tubes in a -20℃ refrigerator. The frequency and time of administration are similar to Figure 2 The timeline in A is consistent.

[0113] After the mice were anesthetized, blood was collected from the eyeball and collected in a 1.5 mL EP tube. The blood was allowed to stand at room temperature for 1 hour until it naturally coagulated, centrifuged at 4000 rpm for 10 minutes, and the serum was collected in a new EP tube. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20 minutes, and standard wells and sample wells were set up. 50 μL of standards of different concentrations were added to each standard well. 10 μL of the sample to be tested was first added to the sample well, and then 40 μL of the sample diluent was added; no addition was made to the blank well. In addition to the blank well, 100 μL of the detection antibody labeled with horseradish peroxidase was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing film, and incubated in a 37°C incubator for 60 minutes. The liquid was discarded, and the well was patted dry on absorbent paper. The washing solution was filled in each well, and it was allowed to stand for 1 minute. The washing solution was shaken off and the well was patted dry on absorbent paper. The washing plate was washed 5 times. 50 μL of substrate A and B were added to each well, and incubated at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value, and then multiply it by 5 to obtain the actual IgE concentration. The results showed that compared with the normal control group mice, the serum IgE level and histamine level of AR mice were significantly increased, indicating that the body had a strong allergic immune response, and the establishment of the AR mouse model was successful. Xiao Chaihu Decoction and a medium-dose Chinese medicine compound (licorice, ginseng, ginger, the same dose as Xiao Chaihu Decoction) significantly inhibited the increase in IgE and histamine levels in AR mice ( Figure 2 B, C), has a strong inhibitory effect on allergic reactions caused by the sensitizer OVA, and also has an inhibitory effect on mast cell stability and histamine release, which helps to reduce the inflammatory response and allergic symptoms caused by histamine. This shows that the main ingredients in Xiao Chaihu Decoction that inhibit allergic rhinitis are licorice, ginseng and ginger, and the inhibitory effect of the Chinese medicine compound on the serum IgE and histamine levels of AR mice is concentration-dependent. Compared with the normal control group, the number of sneezing and scratching of the nose in the AR group mice increased significantly, and the number of times in the Xiao Chaihu Decoction group decreased significantly. The Chinese medicine compound group can also achieve the same effect, and it is concentration-dependent, significantly alleviating the symptoms of allergic rhinitis ( Figure 2 D, E). Subsequent experiments on the mechanism of action used the dose of the medium-dose group.

[0114] Example 3. HE staining to evaluate pathological changes in mouse nasal tissue

[0115] HE staining was used to observe the changes in nasal tissue morphology and nasal mucosal thickness of mice, and to evaluate the effect of the Chinese medicine compound on the nasal mucosa of mice with allergic rhinitis. The tissue sections were first dewaxed, and the sections were immersed in xylene I and II for 10 minutes each; then hydrated, and the sections were immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 1 minute each, and then rinsed with double distilled water for 2 minutes; then stained, the sections were immersed in hematoxylin stain for 1 minute, gently rinsed with tap water for 2 minutes, rinsed with running water for 1 minute, and then separated with 1% hydrochloric acid alcohol for 2 seconds, rinsed with running water for 5 minutes, and finally stained with eosin for 10 seconds and rinsed with running water for 1 minute; then dehydrated and transparentized, the sections were immersed in 75% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 1 minute each, and then immersed in xylene I and II for 2 minutes each; finally, the sections were sealed with neutral resin, observed under a microscope and images were collected. The results showed that compared with the normal control group, the nasal epithelium of the AR group mice was thickened and the olfactory cilia were severely lost. The Chinese herbal compound licorice, ginseng and ginger could significantly inhibit the thickening of the nasal epithelium ( Figure 3 ).

[0116] Example 4. Immunofluorescence staining to assess the integrity of mouse nasal epithelium

[0117] Immunofluorescence staining was used to detect the expression levels of tight junction protein Occludin and cytoplasmic tight adhesion protein ZO-1 in mouse nasal mucosa, and to evaluate the integrity of mouse nasal epithelium. First, paraffin sections were baked at 60℃ for 1.5h to promote tissue adhesion; dewaxed and hydrated with xylene and gradient ethanol (100%, 95%, 85%) in sequence, each step for 5min, and washed with distilled water; antigen retrieval was performed with sodium citrate, and the sections were naturally cooled after heating in a high-temperature microwave oven, and washed in PBS for 3 times, each time for 5min; blocked with 3% BSA for 30min to prevent nonspecific binding; primary antibody was added and incubated at 4℃ overnight; washed with PBS 3 times, each time for 5min; secondary antibody was added and incubated at room temperature in the dark for 1h; washed with PBS 3 times, each time for 5min; nuclear staining reagent DAPI was added and incubated in the dark for 10min; washed with PBS 3 times, each time for 5min; anti-fluorescence quenching sealing agent was used to seal the sections, and the images were observed and collected using a fluorescence microscope. The results showed that compared with the normal control group, the expression of occludin and ZO-1 proteins in the AR group mice was significantly reduced, and the integrity of the nasal epithelium was damaged. The Chinese herbal compound licorice, ginseng and ginger significantly increased the expression levels of the two proteins, which helped to restore the integrity of the nasal epithelium ( Figure 4 ).

[0118] Example 5. PAS staining to assess the level of mouse nasal mucosal goblet cells

[0119] PAS staining was used to detect the number of goblet cells in the nasal mucosa of mice and evaluate the abnormal mucus secretion function of mice. After the sections were dewaxed and hydrated, the periodic acid solution was taken out and balanced to room temperature. 100 μl of periodic acid solution was added to each sample and reacted in the dark for 5 minutes; the periodic acid solution was removed, soaked in distilled water, and washed on a shaker for 5 minutes; 100 μL of Schiff reagent was added, placed in a wet box, and stained in a 37°C incubator in the dark for 5 minutes; after removing the staining solution, soaked in distilled water, and washed on a shaker for 5 minutes; treated with gradient ethanol and xylene, and after proper drying, the sections were sealed with neutral gum; observed and collected images under a microscope. The results showed that compared with the normal control group, the number of goblet cells in the AR group mice increased significantly, resulting in abnormal secretion of mucus in the nasal mucosa of mice, and the number of goblet cells on the nasal mucosa of mice in the Chinese medicine compound treatment group decreased significantly, reflecting that the Chinese medicine compound licorice, ginseng and ginger can improve the abnormal mucus secretion function of AR mice ( Figure 5 ).

[0120] Example 6. Giemsa staining to assess the level of mast cells in mouse nasal mucosa

[0121] Giemsa staining was used to detect the number of mast cells in the nasal mucosa of mice and to evaluate the level of inflammation in the nasal mucosa of mice. After dewaxing and hydration, the slices were added with 200 μl of Giemsa working solution and stained for 1 min; rinsed with distilled water from one side and washed thoroughly; treated with gradient ethanol and xylene, dried and sealed with neutral gum, and observed and collected images under a microscope. The results showed that compared with the normal control group, the number of mast cells in the nasal mucosa of mice in the AR group increased significantly, which could cause inflammatory reactions, and the number of mast cells in the Chinese medicine compound treatment group decreased significantly ( Figure 6 ).

[0122] Example 7. Calculation of spleen index of mice with allergic rhinitis

[0123] During the culture period of establishing the allergic rhinitis mouse model, the weight of the mice was recorded every 7 days. After the model was established and the drug administration was completed, the mice were killed and the spleen was removed. The surface moisture of the spleen was absorbed with filter paper and then weighed. The spleen index was calculated using the final body weight and spleen weight: spleen index = spleen weight (g) / body weight (g) to evaluate the immune response level of the mice. The results showed that compared with the normal control group, the spleen of mice with allergic rhinitis was significantly enlarged, indicating that their immune system was in an active state, and the immune cells in the spleen proliferated massively, reflecting the enhanced systemic immune response to allergens. The Chinese herbal compound significantly inhibited the enlargement of the spleen, reduced the excessive proliferation and activation of immune cells in the spleen, and effectively regulated the immune system ( Figure 7A). Compared with the normal control group, the spleen index of mice in the AR group was significantly increased, reflecting the overactivation of the immune system. The spleen index of mice in the TCM compound treatment group was significantly reduced, indicating that the TCM compound licorice, ginseng and ginger have a good effect in regulating immune response and relieving allergic inflammation ( Figure 7 B).

[0124] Example 8. ELISA kit to detect the levels of IL-4, IL-5 and IL-13 in mouse serum

[0125] ELISA kits were used to detect the levels of IL-4, IL-5 and IL-13 in mouse serum and to evaluate the level of nasal mucosal inflammation in mice. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20 minutes, and standard wells and sample wells were set. 50 μL of standard wells with different concentrations were added. 10 μL of the sample to be tested was first added to the sample well, and then 40 μL of sample diluent was added; no addition was made to the blank well. Except for the blank well, 100 μL of detection antibody labeled with horseradish peroxidase was added to each well of the standard well and sample well, and the reaction wells were sealed with a sealing film and incubated in a 37°C incubator for 60 minutes. The liquid was discarded, patted dry on absorbent paper, and each well was filled with washing solution, left to stand for 1 minute, the washing solution was shaken off, and patted dry on absorbent paper. The washing plate was washed 5 times. 50 μL of substrate A and B were added to each well, and incubated at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value and then multiply it by 5 to obtain the actual concentration of IL-4, IL-5 and IL-13. IL-4, IL-5 and IL-13 in allergic rhinitis promote IgE synthesis, eosinophil activation and mucosal inflammation, leading to the occurrence and aggravation of allergic symptoms. The results showed that compared with the normal control group, the levels of serum IL-4, IL-5 and IL-13 in AR group mice were significantly increased, indicating that the Th2 immune response in mice was enhanced, which would promote IgE-mediated allergic reactions, promote the proliferation and activation of eosinophils, promote the secretion of nasal mucosal mucus, and aggravate the allergic reactions of AR mice. The Chinese herbal compound can significantly inhibit the levels of IL-4, IL-5 and IL-13, reduce the damage of inflammatory mediators to tissues, inhibit the secretion of mucus, and relieve allergic symptoms such as nasal congestion and runny nose, indicating that the Chinese herbal compound licorice, ginseng and ginger have a positive effect in relieving allergic inflammation and improving symptoms ( Figure 8 A, B, C).

[0126] Example 9. HE staining to evaluate the effect of Chinese herbal compound on liver and kidney tissues of mice

[0127] HE staining was used to detect the pathological structure of mouse liver and kidney tissues, and to evaluate the toxicity of the Chinese medicine compound on the liver and kidneys of mice. The tissue sections were first dewaxed, and the sections were immersed in xylene I and II for 10 minutes each; then hydrated, and the sections were immersed in anhydrous ethanol, 95% ethanol, 80% ethanol, and 75% ethanol for 1 minute each, and then rinsed with double distilled water for 2 minutes; then stained, the sections were immersed in hematoxylin stain for 1 minute, gently rinsed with tap water for 2 minutes, rinsed with running water for 1 minute, and then separated with 1% hydrochloric acid alcohol for 2 seconds, rinsed with running water for 5 minutes, and finally stained with eosin for 10 seconds and rinsed with running water for 1 minute; then dehydrated and transparentized, the sections were immersed in 75% ethanol, 80% ethanol, 95% ethanol and anhydrous ethanol for 1 minute each, and then immersed in xylene I and II for 2 minutes each; finally, the sections were sealed with neutral resin, and observed and collected images under a microscope. The results of liver tissue HE staining showed that in the liver tissue of AR mice, the hepatic plates were arranged regularly and the hepatic lobule structure was not disordered; in the mice in the Chinese medicine compound administration group, the hepatic plates were also regular and the hepatic lobule structure was complete. Some hepatocytes showed binucleation, indicating that the liver function was in an active state, which may be related to the increased metabolic activity of hepatocytes under the action of drugs ( Fig. 9 A). The results of HE staining of renal tissue showed that there were no obvious abnormalities in the glomeruli and surrounding structures of AR mice and mice in the Chinese medicine compound administration group, and no pathological changes were found in the renal tubules and interstitium, indicating that the Chinese medicine compound licorice, ginseng and ginger had no significant effect on the tissue structure of the kidney ( Fig. 9 B).

[0128] Example 10. ELISA kit to detect the level of liver function and kidney function in mice

[0129] ELISA kits were used to detect the levels of serum alanine aminotransferase ALT and urea nitrogen BUN in mice to evaluate the toxicity of the Chinese medicine compound on the liver and kidney of mice. ALT detection: pre-warm the matrix solution to 37°C; add 20μL matrix solution and 5μL sample to be tested in the measurement well, and add 20μL matrix solution to the control well; gently shake the microplate to mix, and react at 37°C for 30min; add 20μL 2,4-dinitrophenylhydrazine solution to the measurement well, and add 5μL 2,4-dinitrophenylhydrazine solution to the control well; gently shake the microplate to mix, and react at 37°C for 20min; add 200μL 0.4 mol / L sodium hydroxide solution to each measurement well and control well; gently shake the microplate to mix, and place at room temperature for 15min; set the wavelength to 505nm, measure the OD value of each well, and subtract the OD value of the control well from the OD value of the measurement well to obtain the net OD value; substitute the standard curve to calculate the ALT activity value. BUN detection: add 20 μL of distilled water to the blank tube, add 20 μL of 10 mmol / L BUN standard application solution to the standard tube, and add 20 μL of the sample to be tested to the measurement tube; add 250 μL of buffered enzyme solution to each tube; mix well and incubate in a 37°C constant temperature box for 10 minutes; add 1 mL of color developer and 1 mL of alkaline sodium hypochlorite to each tube; mix well and incubate in a 37°C constant temperature box again for 10 minutes; set the detection wavelength to 640 nm, the optical path to 1 cm, adjust to zero with distilled water, measure the absorbance OD value of each tube, and substitute it into the formula to obtain the urea nitrogen concentration: urea nitrogen concentration (mmol / L) = (A 测定 -A 空白 ) / (A 标准 -A 空白 )×C 标准。 The results of liver function tests showed that the ALT levels of AR mice and mice in the Chinese medicine compound administration group were within 15U / L, which was within the normal range of liver function, indicating that the Chinese medicine compound licorice, ginseng and ginger had no abnormal effects on liver function. The results of kidney function tests showed that the BUN levels of AR mice and mice in the Chinese medicine compound administration group were within 10mmol / L, which was within the normal range of kidney function, indicating that the Chinese medicine compound licorice, ginseng and ginger had no abnormal effects on the kidneys (Table 1).

[0130] Table 1. Levels of liver alanine aminotransferase and kidney urea nitrogen

[0131]

[0132] CTL: normal control group mice AR: allergic rhinitis mice TCM: Chinese medicine compound licorice, ginseng and ginger treatment group mice.

[0133] Example 11. Animal behavior experiment to evaluate the effects of other components of Xiao Chaihu Decoction on mice with allergic rhinitis

[0134] In order to further verify that the Chinese herbal compound (licorice, ginseng, and ginger) is the key component of Xiao Chaihu Decoction for the treatment of allergic rhinitis, this experiment also tested the effects of other components in Xiao Chaihu Decoction on mice with allergic rhinitis.

[0135] Eight-week-old male BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. On days 1, 8, and 15, 200 μl of PBS containing 25 μg of sensitizer chicken egg albumin OVA and 2 mg of adjuvant aluminum hydroxide were intraperitoneally injected. On days 22-35, 20 μl of PBS containing 25 mg / mL OVA was intranasally infused. After the last nasal challenge on day 35, the number of nose scratching and sneezing of the mice within 20 min was immediately observed and recorded, and the scores were given. Sneezing: 1-3, 1 point; 4-10, 2 points; 11 or more, 3 points. Scratching the nose: 1 point for gently rubbing the nose several times; 2 points for frequent nose scratching; 3 points for continuous nose scratching with both forelimbs. The above two symptom scores were added together, and a total score greater than 5 points indicated that the model was successfully established.

[0136] The experiment was divided into 4 groups: normal control group, allergic rhinitis mouse group, Xiao Chaihu Decoction other components (Bupleurum, Scutellaria, Pinellia and Jujube) treatment group and loratadine positive control group. Each group had 20 mice, which were divided into 2 cages, with 10 mice in each cage. The dosage of other components of Xiao Chaihu Decoction was: Bupleurum 15g, Scutellaria 9g, Pinellia 9g and Jujube 6g. The preparation method was as follows: (1) Soak in cold water: Put the medicinal materials into an appropriate amount of cold water, making sure that the water volume is enough to cover the medicinal materials by 2-5cm to prevent the medicinal materials from being boiled dry or burned. The soaking time was 40min. (2) Boil over high heat: Put the soaked medicinal materials into a casserole, add 300mL of water, and boil over high heat. (3) Boil over low heat: After the medicinal liquid boils, turn to low heat and continue to boil. Start the timer at this time and boil for 30min. During the boiling process, it is necessary to stir continuously with a glass rod to ensure that the medicinal materials are fully boiled. (4) Filter the medicinal solution: After boiling for 30 minutes, use a sieve to filter the medicinal solution and pour it into a beaker. Put the filter residue back into the casserole, add 300 mL of water, boil again, and then simmer for 30 minutes on low heat. During this process, it is also necessary to stir continuously. (5) Mix the medicinal solution: Filter the medicinal solution from the second boiling, combine it with the medicinal solution from the first boiling, and pour it into the casserole. (6) Evaporation and concentration: Evaporate and concentrate the combined medicinal solution in the casserole. Let the medicinal solution cool, divide it into 15 mL centrifuge tubes, and store it in a -20°C refrigerator. The Chinese medicine compound was administered to mice by gavage on days 1-3, 8-10, 15-17, and 22-35. The dosage was calculated based on the ratio of human and mouse body surface areas, with a conversion coefficient of 9.1 for adults and mice. Based on a 70kg adult taking one dose of medicine per day, the single dosage of mice in the Xiao Chaihu Decoction other component treatment group was 5.07g / kg, and the dosage of the decoction after conversion was 0.2mL. The dosage of loratadine in the positive control group was 1.5mg / kg, and the frequency and time of administration were the same as those in the Chinese medicine compound treatment group.

[0137] After the mice were anesthetized, blood was collected from the eyeball and collected in a 1.5 mL EP tube. The blood was allowed to stand at room temperature for 1 hour until it naturally coagulated, centrifuged at 4000 rpm for 10 minutes, and the serum was collected in a new EP tube. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 20 minutes, and standard wells and sample wells were set up. 50 μL of standards of different concentrations were added to each standard well. 10 μL of the sample to be tested was first added to the sample well, and then 40 μL of the sample diluent was added; no addition was made to the blank well. In addition to the blank well, 100 μL of the detection antibody labeled with horseradish peroxidase was added to each well of the standard well and the sample well, and the reaction well was sealed with a sealing film, and incubated in a 37°C incubator for 60 minutes. The liquid was discarded, and the well was patted dry on absorbent paper. The washing solution was filled in each well, and it was allowed to stand for 1 minute. The washing solution was shaken off and the well was patted dry on absorbent paper. The washing plate was washed 5 times. 50 μL of substrate A and B were added to each well, and incubated at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 minutes, and draw a standard curve to obtain the curve equation. Substitute the OD value of each sample into the equation to obtain the corresponding value, and then multiply it by 5 to obtain the actual IgE concentration. The results showed that compared with the normal control group mice, the serum IgE level and histamine level of AR mice were significantly increased, indicating that the body had a strong allergic immune response, and the establishment of the AR mouse model was successful. The treatment group with other components of Xiao Chaihu Decoction (Bupleurum, Scutellaria, Pinellia and Jujube) failed to significantly inhibit the increase of IgE and histamine levels in AR mice ( Fig.10 A, B), indicating that the other components of Xiao Chaihu Decoction have no inhibitory effect on the allergic reaction caused by the sensitizer OVA. Compared with the normal control group, the number of sneezing and scratching of the nose in the AR group mice increased significantly, and the number of sneezing and scratching of the nose in the group treated with other components of Xiao Chaihu Decoction did not decrease significantly, indicating that the other components of Xiao Chaihu Decoction cannot relieve the symptoms of allergic rhinitis ( Fig.10 C, D).

[0138] The above is only a preferred embodiment of the present invention. It should be noted that the embodiments of the present invention are not limited to the embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the protection scope of the present invention.

Claims

1. A Chinese medicine composition for preventing and / or treating allergic rhinitis, prepared from the following raw materials in weight ratio: 4.5-18 weight portions of licorice root 4.5-18 parts by weight of ginseng 4.5-18 parts by weight of ginger.

2. The Chinese medicine composition according to claim 1, characterized in that: Prepared from the following raw materials in the following weight ratio: 9 parts by weight of licorice root Ginseng 9 parts by weight 9 parts by weight of ginger.

3. The Chinese medicine composition according to claim 1, characterized in that The invention can be prepared into any pharmaceutically acceptable dosage form, including oral and external dosage forms.

4. The Chinese medicine composition according to claim 1, characterized in that: Tablets, capsules, lozenges, granules, pills, powders, pastes, pills, suspensions, solutions, injections, suppositories, ointments, sprays, drops, pellets or patches.

5. The Chinese medicine composition according to claim 1, characterized in that: When preparing the medicament, a suitable pharmaceutically acceptable carrier can be selectively added, and the pharmaceutically acceptable carrier is selected from the group consisting of mannitol, sorbitol, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate, cysteine ​​hydrochloride, thioglycolic acid, methionine, vitamin C, disodium EDTA, sodium calcium EDTA, carbonates, acetates, phosphates of monovalent alkali metals or their aqueous solutions, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, alginate, gelatin, polyvinyl pyrrolidone, glycerol, soil temperature 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, β-cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate, magnesium stearate, etc.

6. Use of the Chinese medicine composition according to claim 1 in the preparation of medicines for preventing and treating allergic rhinitis.

7. The use according to claim 6, characterized in that: The Chinese medicine composition can significantly inhibit the number of sneezing and nose scratching in mice with allergic rhinitis, and significantly inhibit the levels of IgE and histamine in the serum of mice with allergic rhinitis.

8. The use according to claim 6, characterized in that: The Chinese medicine composition can significantly reduce the thickening of nasal mucosa caused by allergic rhinitis and increase the expression of nasal mucosal tight junction protein Occludin and cytoplasmic tight adhesion protein ZO-1. Occludin and ZO-1 play an important role in maintaining the integrity of nasal epithelial cells, regulating permeability and participating in cell signal transduction.

9. The use according to claim 6, characterized in that: The Chinese medicine composition can reduce the number of goblet cells and mast cells on the nasal mucosa and inhibit the levels of inflammatory factors IL-4, IL-5 and IL-13 in serum.

10. The method for preparing the Chinese medicine composition according to claim 1, characterized in that: The following steps are involved: (1) Soaking in cold water: Place the herbs in an appropriate amount of cold water, making sure that the water level is enough to cover the herbs by 2-5 cm to prevent them from drying out or burning. The soaking time is 20-60 minutes. (2) Boil over high heat: Put the soaked herbs into a casserole, add 150-600 mL of water, and boil over high heat; (3) Boil over low heat: After the liquid medicine boils, turn to low heat and continue boiling. Start timing at this time and boil for 15-60 minutes. (4) Filtering the liquid medicine: After boiling for 30 minutes, filter the liquid medicine through a sieve and pour it into a beaker. Put the residue back into the casserole, add 150-600 mL of water, boil again, and then boil over low heat for 15-60 minutes. (5) Mixing the medicinal liquid: Filter out the medicinal liquid from the second boiling, combine it with the medicinal liquid from the first boiling, and pour it into a casserole; (6) Evaporation and concentration: The combined medicinal solution is evaporated and concentrated in a casserole to obtain the product.

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