Application of panoxadiol in prevention and treatment of allergic rhinitis

Abstract

The invention aims to provide application of panoxadiol in preparation of a medicine for treating and / or preventing allergic rhinitis. The effective component panoxadiol of the classic famous formula Xiaochaihu decoction for treating allergic rhinitis is screened out through pharmacology.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of ginsenoside in preventing and treating allergic rhinitis. Background Art

[0002] Allergic rhinitis (AR) is a disease that is widely prevalent around the world. It is an inflammatory disease of the nasal mucosa caused by allergen stimulation and IgE-mediated release of histamine as the main inflammatory mediator. It is characterized by clinical symptoms such as 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. The current methods for treating allergic rhinitis are mainly to stay away from allergens and use drugs. The main therapeutic drugs include steroid hormones, antihistamines, mast cell membrane stabilizers and nasal spray decongestants. The drugs currently used in clinical practice can generally only control the symptoms of allergic rhinitis, cannot achieve the purpose of radical cure, and have many adverse reactions.

[0003] Many Chinese herbal compound prescriptions are also used in the treatment of allergic rhinitis. In the treatment system of traditional Chinese medicine, Chinese herbal compound prescriptions, due to their multi-component and multi-target characteristics, can often achieve the effects of overall conditioning and treating both the symptoms and the root causes through multi-level and multi-pathway mechanisms of action. This complex mode of action is not only the advantage of Chinese medicine, but also provides the basis for its unique efficacy in the treatment of diseases. However, this complexity of multiple components also brings challenges. The relationship between the ingredients in the prescription is intricate, and there may be synergistic or antagonistic effects between the ingredients, making it difficult to accurately analyze the efficacy mechanism, especially in the context of modern medicine requiring clear ingredients and mechanisms of action. This has become an obstacle to the standardization and internationalization of Chinese medicine.

[0004] In order to solve this problem, the present invention applies the screening technology of modern pharmacology to classic prescriptions, screens the key effective ingredients in the prescriptions through network pharmacology, and analyzes their independent mechanisms of action. In previous studies, we found that the classic prescription Xiao Chaihu Decoction has a significant inhibitory effect on allergic rhinitis. However, Xiao Chaihu Decoction includes a variety of Chinese medicinal materials such as Bupleurum, Scutellaria, Pinellia, Glycyrrhiza, Ginseng, Ginger, and Jujube, with many ingredients and complex interactions. In order to clarify its pharmacodynamic mechanism and promote its application, we further screened out the key compound ginsenodiol in Xiao Chaihu Decoction. Its pharmacological mechanism is gradually analyzed from prescriptions to single compounds, and on this basis, the development path of innovative drugs is explored.

[0005] Panaxadiol, as an important saponin active ingredient in ginseng, has shown great potential in the research of various inflammatory diseases. Since allergic rhinitis is essentially a chronic inflammatory disease mediated by immunoglobulin IgE, its pathological process involves immune disorders, release of inflammatory mediators and uncontrolled cell proliferation. Panaxadiol is expected to play an important role in the treatment of AR through its immunomodulatory and anti-inflammatory properties. The research results of this project will not only provide a scientific basis for the efficacy of ginseng, but also help promote the modernization and internationalization of traditional Chinese medicine, so that classic prescriptions can be more widely recognized and applied in the global medical system. Summary of the invention

[0006] The object of the present invention is to provide the use of ginsenoside in preparing a medicine for treating and or preventing allergic rhinitis.

[0007] The present invention screens out ginsenoside, an effective ingredient of the classic prescription Xiao Chaihu Decoction for treating allergic rhinitis, through network pharmacology, and further verifies the application of ginsenoside in preventing and treating allergic rhinitis through experiments.

[0008] The pharmaceutical application of the present invention refers to:

[0009] Panaxadiol 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.

[0010] Panaxadiol 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. Restoring the normal levels of these two proteins is crucial for protecting the nasal mucosa from allergens and pathogens, helping to repair the epithelial barrier and alleviate symptoms.

[0011] Panaxadiol can significantly reduce the number of goblet cells and mast cells in the nasal mucosa and inhibit the levels of inflammatory factors IL-4, IL-5 and IL-13 in serum.

[0012] Panaxadiol had no significant effect on the tissue structure of the liver and kidneys. The expression levels of alanine aminotransferase (ALT) and urea nitrogen (BUN) in serum also confirmed that panaxadiol had no significant effect on liver and kidney function.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] The effective ingredients and drug efficacy of the present invention are obtained through a large number of experimental screenings:

[0023] 1. Screening of the active ingredients of Xiao Chaihu Decoction for the treatment of allergic rhinitis using network pharmacology analysis

[0024] The TCMSP database (https: / / old.tcmsp-e.com) was used to search for the main components of all drugs in Xiao Chai Hu Tang, and the active ingredients of Xiao Chai Hu Tang were filtered and screened according to bioavailability ≥ 30% and drug-likeness ≥ 0.18. The Swiss Target Prediction (http: / / swisstargetprediction.ch / ) database was used to further evaluate the potential therapeutic targets of the active ingredients based on pharmacokinetics (GI absorption: high) and drug-likeness (Lipinski's five rules and Ghose's law).

[0025] The genes related to allergic rhinitis were screened using the keyword "allergic rhinitis" in the Genecard database (http: / / www.genecards.org), the OMIM database (http: / / omim.org / ), and the DisGeNET database (http: / / www.disgenet.org / web / DisGeNET / ).

[0026] The mapping tool Venny 2.1 was used to construct the intersection of potential therapeutic targets of Xiao Chaihu Decoction and genes related to allergic rhinitis, which were the key genes for Xiao Chaihu Decoction to treat allergic rhinitis. The information interaction network of the targets in the intersection was constructed using the STRING database, and the properties of the network were analyzed using Cytoscape 3.9.1 software. Clustering and degree analysis were performed, and the top ten core targets were screened out based on Degree, Betweenness, and Closeness. Cytoscape was continued to be used to search for the corresponding active ingredients in Xiao Chaihu Decoction based on the core targets, and a target-drug component network diagram was constructed. According to the Degree value, ginsenodiol was screened out, which may have great potential for the treatment of allergic rhinitis. The three-dimensional structure of ginsenodiol was obtained from the compound information database PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and the binding activity of ginsenodiol with the core targets was further verified by molecular docking using AutoDock Vina and PyMOL software.

[0027] 2. Construction of allergic rhinitis mouse model and drug administration

[0028] 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.

[0029] Establishment of a mouse model of allergic rhinitis

[0030] 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 provocation on day 35, the mice were immediately observed and scored for the number of nose scratching, sneezing, and runny nose within 30 minutes. Sneezing: 1 point for 1-3, 2 points for 4-10, and 3 points for more than 11. 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 that the model was successfully established. The model establishment process is as follows: Figure 2 Show.

[0031] Grouping and dosing

[0032] The experimental groups were: normal control group, allergic rhinitis mouse group, Xiao Chaihu Decoction treatment group, ginsenodiol 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 Xiao Chaihu Decoction was: 15g of Bupleurum, 9g of Scutellaria, 9g of Pinellia, 9g of Radix Glycyrrhizae, 9g of Ginseng Slices, 9g of Ginger, and 6g of Jujube. 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 timing 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 filtrate 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 dosage is converted according to the ratio of human to mouse body surface area. The conversion coefficient between adults and mice is 9.1. Based on one dose of medicine for a 70 kg adult per day, the single dose for mice is 8.58 g / kg, and the volume of the combined water decoction is 0.2 mL. Panaxadiol was divided into high, medium (calculated based on the amount of panaxadiol contained in 9g of ginseng) and low dose groups, with the dosages of 10mg / kg, 5mg / kg and 2.5mg / kg respectively. The dosage of loratadine in the positive control group was 1.5mg / kg. The mice were given drugs by gavage on days 1-3, 8-10, 15-17 and 22-35.

[0033] ELISA kit to detect mouse serum IgE and histamine levels

[0034] 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.

[0035] 3. HE staining to evaluate pathological changes in mouse nasal tissue

[0036] HE staining was used to observe the changes in nasal tissue morphology and nasal mucosal thickness of mice, and to evaluate the effect of ginsenoside on the nasal mucosa of mice with allergic rhinitis. 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, 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, sealed with neutral resin, observed under a microscope and collected images.

[0037] 4. Immunofluorescence staining to assess the integrity of mouse nasal epithelium

[0038] 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.

[0039] 5. PAS staining to evaluate the level of mouse nasal mucosal goblet cells

[0040] 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.

[0041] 6. Giemsa staining to assess the level of mast cells in mouse nasal mucosa

[0042] 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; they were thoroughly rinsed with distilled water from one side; they were treated with gradient ethanol and xylene, dried, and sealed with neutral gum, and observed and collected images under a microscope.

[0043] 7. Calculation of spleen index of mice with allergic rhinitis

[0044] 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.

[0045] 8. ELISA kit to detect the levels of IL-4, IL-5 and IL-13 in mouse serum

[0046] 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.

[0047] 9. HE staining to evaluate the effects of ginsenoside on mouse liver and kidney tissues

[0048] HE staining was used to evaluate the toxicity of panaxadiol to 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, 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, sealed with neutral resin, observed under a microscope and collected images.

[0049] 10. ELISA kit to detect the liver and kidney function levels of mice

[0050] ELISA kits were used to detect the levels of serum alanine aminotransferase ALT and urea nitrogen BUN in mice to evaluate the toxicity of ginsenosides on the liver and kidneys 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。

[0051] 11. Statistical analysis

[0052] 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.

[0053] Explanation of some English abbreviations or terms in the manual:

[0054] IgE: Immunoglobulin E

[0055] Occludin: tight junction protein

[0056] ZO-1: cytoplasmic tight adhesion protein

[0057] Degree: degree

[0058] Betweenness

[0059] Closeness: Closeness

[0060] OVA: ovalbumin

[0061] ALT: Alanine transaminase

[0062] BUN: urea nitrogen

[0063] IL-4: interleukin 4

[0064] IL-5: interleukin 5

[0065] IL-13: Interleukin 13 BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 Network pharmacology screening of the active ingredients of Xiao Chaihu Decoction for the treatment of allergic rhinitisA. Intersection of potential therapeutic targets of Xiao Chaihu Decoction and genes related to allergic rhinitisB. Screening of core targetsC. Information interaction network of targetsD. Target-drug ingredient network diagramE. Molecular docking of ginsenodiol with core targetsXCHT: Xiao Chaihu DecoctionAR: Allergic rhinitisPD: Ginsenodiol

[0067] 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 treatment group miceHPD: high-dose ginsenodiol treatment group miceMPD: medium-dose ginsenodiol treatment group miceLPD: low-dose ginsenodiol treatment group miceLor: loratadine positive control group mice*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0068] Figure 3. HE staining was used to evaluate the pathological changes of mouse nasal tissueA. Nasal tissue morphologyB. Nasal mucosal thicknessCTL: normal control group miceAR: allergic rhinitis micePD: ginsenodiol-treated group miceLor: loratadine positive control group mice****P<0.0001.

[0069] 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 CTL: normal control mice AR: allergic rhinitis mice PD: panaxadiol-treated mice Lor: loratadine-positive control mice

[0070] Figure 5. PAS staining to evaluate the level of goblet cells in the nasal mucosa of mice CTL: normal control mice AR: allergic rhinitis mice PD: panaxadiol-treated mice Lor: loratadine-positive control mice

[0071] Figure 6. Evaluation of the level of mast cells in the mouse nasal mucosa by Giemsa staining CTL: normal control mice AR: allergic rhinitis mice PD: panaxadiol-treated mice Lor: loratadine-positive control mice

[0072] Figure 7. Spleen index of mice with allergic rhinitis A. Mouse spleen size B. Spleen index CTL: Normal control group mice AR: Allergic rhinitis mice PD: Panaxadiol-treated group mice Lor: Loratadine positive control group mice ****P < 0.0001.

[0073] Figure 8. Levels of IL-4, IL-5 and IL-13 in mouse serumA. Levels of IL-4 in serumB. Levels of IL-5 in serumC. Levels of IL-13 in serumCTL: mice in normal control groupAR: mice with allergic rhinitisPD: mice in the panaxadiol-treated groupLor: mice in the loratadine positive control group*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0074] Figure 9. HE staining was used to evaluate the effect of ginsenodiol on liver and kidney tissues of mice. A. HE staining of liver tissue B. HE staining of kidney tissue CTL: mice in normal control group AR: mice with allergic rhinitis PD: mice in ginsenodiol treatment group

[0075] Fig.10 .Animal behavioral experiment evaluates the effect of 4,6-dioxoheptanoic acid, another component of ginseng, on mice with allergic rhinitisA. Serum IgE levelB. Serum histamine levelC. Number of sneezesD. Number of nose scratchingCTL: normal control group miceAR: allergic rhinitis micesuchilactone: 4,6-dioxoheptanoic acid treated miceLor: loratadine positive control group mice****P<0.0001.

[0076] Fig.11 .Experimental technology roadmap. DETAILED DESCRIPTION

[0077] The present invention is further illustrated by the following examples.

[0078] 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.

[0079] Example 1. Screening of the effective ingredients of Xiao Chaihu Decoction for the treatment of allergic rhinitis using network pharmacology

[0080] The TCMSP database (https: / / old.tcmsp-e.com) was used to search for the main components of all drugs in Xiao Chai Hu Tang, and the active ingredients of Xiao Chai Hu Tang were filtered and screened according to bioavailability ≥ 30% and drug-likeness ≥ 0.18. The Swiss Target Prediction (http: / / swisstargetprediction.ch / ) database was used to further evaluate the potential therapeutic targets of the active ingredients based on pharmacokinetics (GI absorption: high) and drug-likeness (Lipinski's five rules and Ghose's law).

[0081] The genes related to allergic rhinitis were screened using the keyword "allergic rhinitis" in the Genecard database (http: / / www.genecards.org), the OMIM database (http: / / omim.org / ), and the DisGeNET database (http: / / www.disgenet.org / web / DisGeNET / ).

[0082] The mapping tool Venny 2.1 was used to construct the intersection of potential therapeutic targets of Xiao Chaihu Decoction and genes related to allergic rhinitis, which were the key genes for Xiao Chaihu Decoction to treat allergic rhinitis. The information interaction network of the targets in the intersection was constructed using the STRING database, and the properties of the network were analyzed using Cytoscape 3.9.1 software. Clustering, degree value and other analyses were performed, and the top ten core targets were screened out according to Degree, Betweenness and Closeness. Cytoscape was continued to be used to search for the corresponding active ingredients in Xiao Chaihu Decoction according to the core targets, and a target-drug component network diagram was constructed. The active ingredients with great potential for the treatment of allergic rhinitis were screened according to the Degree value. The three-dimensional structure of the active ingredient was further obtained from the compound information database PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and the binding activity of the active ingredient with the core target was verified by molecular docking using AutoDock Vina and PyMOL software.

[0083] The results showed that 1102 potential therapeutic targets of Xiao Chaihu Tang active ingredients were screened in the TCMSP database and Swiss Target Prediction database based on pharmacokinetics and drug-like properties. 1339 target genes related to allergic rhinitis were screened in the Genecard database, OMIM database, and DisGeNET database using the keyword "allergic rhinitis". The intersection of potential therapeutic targets of Xiao Chaihu Tang and genes related to allergic rhinitis was constructed, and 202 common genes were screened ( Figure 1A ). The information interaction network of the targets in the intersection was constructed using the STRING database, and the top ten core targets were screened out according to Degree, Betweenness and Closeness, including Bcl2, AKT1, NFκB1, IL1B, TNF, IL6, PPARG, EGFR, STAT3 and MMP9 ( Figure 1B , C). Use Cytoscape to construct a target-drug component network diagram ( Figure 1D ), and the active ingredient with a higher score was selected according to the Degree value, which was ginsenodiol. The binding activity of ginsenodiol with the core target was further verified by molecular docking. Its binding energy with the core target was low and it had a strong binding activity, indicating that ginsenodiol has great potential for the treatment of allergic rhinitis ( Figure 1E ).

[0084] Example 2. Establishment of allergic rhinitis mouse model and drug administration

[0085] 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 2A shown.

[0086] The experimental groups were: normal control group, allergic rhinitis mouse group, Xiao Chaihu Decoction treatment group, ginsenodiol 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 Xiao Chaihu Decoction was: 15g of Bupleurum, 9g of Scutellaria, 9g of Pinellia, 9g of Radix Glycyrrhizae, 9g of Ginseng Slices, 9g of Ginger, and 6g of Jujube. 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 timing 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 filtrate 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 dosage is converted according to the ratio of human to mouse body surface area. The conversion coefficient between adults and mice is 9.1. Based on one dose of medicine for a 70 kg adult per day, the single dose for mice is 8.58 g / kg, and the volume of the combined water decoction is 0.2 mL. Panaxadiol was divided into high, medium (calculated based on the amount of panaxadiol contained in 9g of ginseng) and low dose groups, with the dosages of 10mg / kg, 5mg / kg and 2.5mg / kg respectively. The dosage of loratadine in the positive control group was 1.5mg / kg. The mice were given drugs by gavage on days 1-3, 8-10, 15-17 and 22-35.

[0087] 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 left to stand at room temperature for 1h until it naturally coagulated, centrifuged at 4000rpm for 10min, and the serum was collected into a new EP tube. 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 blank well was added. In addition to the blank well, 100μL of horseradish peroxidase-labeled detection antibody 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℃ 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 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 high and medium doses of ginsenodiol significantly inhibited the increase in IgE and histamine levels in AR mice ( Figure 2B , 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, indicating that ginsenodiol is the main component of Xiao Chaihu Decoction that inhibits allergic rhinitis, and the inhibitory effect of ginsenodiol on serum IgE and histamine levels in AR mice is concentration-dependent. Compared with the normal control group, the number of sneezing and scratching noses in the AR group mice increased significantly, and the number of times in the Xiao Chaihu Decoction group decreased significantly. The ginsenodiol group can also achieve the same effect, and it is concentration-dependent, significantly alleviating the symptoms of allergic rhinitis ( Figure 2D , E). Subsequent experiments on the mechanism of action will use the dose of the high-dose group.

[0088] Example 3. HE staining to evaluate pathological changes in mouse nasal tissue

[0089] HE staining was used to observe the changes in nasal tissue morphology and nasal mucosal thickness of mice, and to evaluate the effect of ginsenoside on the nasal mucosa of mice with allergic rhinitis. 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, 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, sealed with neutral resin, observed under a microscope and collected images. 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. Panaxadiol could significantly inhibit the thickening of the nasal epithelium ( Figure 3) .

[0090] Example 4. Immunofluorescence staining to assess the integrity of mouse nasal epithelium

[0091] 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. Panaxadiol significantly increased the expression levels of the two proteins, which helped to restore the integrity of the nasal epithelium ( Figure 4) .

[0092] Example 5. PAS staining to assess the level of mouse nasal mucosal goblet cells

[0093] 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. 100μl 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; 100μL 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 ginsengdiol-treated group decreased significantly, indicating that ginsengdiol can improve the abnormal mucus secretion function of AR mice ( Figure 5) .

[0094] Example 6. Giemsa staining to assess the level of mast cells in mouse nasal mucosa

[0095] 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 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. 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 ginsengdiol treatment group decreased significantly ( Figure 6) .

[0096] Example 7. Calculation of spleen index of mice with allergic rhinitis

[0097] 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. Panaxadiol 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 ( Fig. 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 ginsenodiol treatment group was significantly reduced, indicating that the drug has a good effect in regulating immune response and relieving allergic inflammation ( Figure 7B ).

[0098] Example 8. ELISA kit to detect the levels of IL-4, IL-5 and IL-13 in mouse serum

[0099] 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. Panaxadiol 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 panaxadiol has a positive effect in relieving allergic inflammation and improving symptoms ( Fig. 8A , B, C).

[0100] Example 9. HE staining to evaluate the effect of ginsenoside on mouse liver and kidney tissues

[0101] HE staining was used to detect the pathological structure of mouse liver and kidney tissues, and to evaluate the toxicity of ginsenodiol to mouse liver and kidney. Tissue sections were first dewaxed, and the sections were immersed in xylene I and II for 10 min each; then hydrated, and the sections were 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, the sections were 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, rinsed with running water for 1 min; then dehydrated and transparentized, the sections were 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. The results of liver tissue HE staining showed that in the liver tissue of AR mice, the liver plates were arranged regularly and the liver lobule structure was not disordered; in the mice in the ginsenodiol group, the liver plates were also regular and the liver lobule structure was complete. Some hepatocytes showed binucleation, indicating that the liver function was active, which may be related to the increased metabolic activity of hepatocytes under the action of the drug ( Fig.9A ). 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 ginsenodiol group, and no pathological changes were found in the renal tubules and interstitium, indicating that ginsenodiol had no significant effect on the tissue structure of the kidney ( Fig. 9B ).

[0102] Example 10. ELISA kit to detect the level of liver function and kidney function in mice

[0103] ELISA kits were used to detect the levels of serum alanine aminotransferase ALT and urea nitrogen BUN in mice to evaluate the toxicity of ginsenosides on the liver and kidneys of mice. ALT detection: pre-warm the matrix solution to 37°C; add 20 μL matrix solution and 5 μL sample to be tested in the assay wells, and add 20 μL matrix solution to the control wells; gently shake the microplate to mix, and react at 37°C for 30 minutes; add 20 μL 2,4-dinitrophenylhydrazine solution to the assay wells, and add 5 μL 2,4-dinitrophenylhydrazine solution to the control wells; gently shake the microplate to mix, and react at 37°C for 20 minutes; add 200 μL 0.4 mol / L sodium hydroxide solution to each assay well and control well; gently shake the microplate to mix, and place at room temperature for 15 minutes; set the wavelength to 505 nm, measure the OD value of each well, and subtract the OD value of the control well from the OD value of the assay 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 ginsenodiol group were within 15U / L, which was within the normal range of liver function, indicating that ginsenodiol had no abnormal effect on liver function. The results of renal function tests showed that the BUN levels of AR mice and mice in the ginsenodiol group were within 10mmol / L, which was within the normal range of renal function, indicating that ginsenodiol had no abnormal effect on the kidneys (Table 1).

[0104] Table 1. Levels of liver alanine aminotransferase and kidney urea nitrogen

[0105]

[0106] CTL: Normal control mice AR: Allergic rhinitis mice PD: Panaxadiol-treated mice

[0107] Example 11. Animal behavior experiment to evaluate the effect of 4,6-dioxoheptanoic acid (suchilactone), another component of ginseng, on mice with allergic rhinitis

[0108] Through network pharmacology, it is also predicted that 4,6-dioxoheptanoic acid (suchilactone) in ginseng has a certain binding activity with the target of allergic rhinitis. This experiment will verify whether 4,6-dioxoheptanoic acid can also effectively relieve the symptoms of allergic rhinitis mice.

[0109] 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.

[0110] The experiment was divided into 4 groups: normal control group, allergic rhinitis mouse group, 4,6-dioxoheptanoic acid treatment group and loratadine positive control group. Each group had 20 mice, divided into 2 cages, 10 mice in each cage. The dosage of 4,6-dioxoheptanoic acid was 15 mg / kg. The dosage of loratadine in the positive control group was 1.5 mg / kg. The mice were given drugs by gavage on days 1-3, 8-10, 15-17, and 22-35.

[0111] 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 left to stand at room temperature for 1h until it naturally coagulated, centrifuged at 4000rpm for 10min, and the serum was collected into a new EP tube. 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 blank well was added. In addition to the blank well, 100μL of horseradish peroxidase-labeled detection antibody 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℃ 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 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 4,6-dioxoheptanoic acid treatment group failed to significantly inhibit the increase in IgE and histamine levels in AR mice ( Fig.10 A, B), indicating that other components of ginseng 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 4,6-dioxoheptanoic acid group did not decrease significantly, indicating that other components of ginseng cannot relieve the symptoms of allergic rhinitis ( Fig.10 C, D).

[0112] The above description 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 scope of protection of the present invention.

Claims

1. Use of ginsenoside in the preparation of drugs for treating and / or preventing allergic rhinitis.

2. The use according to claim 1, characterized in that: The invention discloses an application of ginsenodiol in the preparation of a drug which 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.

3. The use according to claim 1, characterized in that: The application of ginsenodiol in the preparation of a drug for alleviating the thickening of the nasal mucosa caused by allergic rhinitis and increasing the expression of the nasal mucosal tight junction protein Occludin and the cytoplasmic tight adhesion protein ZO-1.

4. The use according to claim 1, characterized in that: The invention discloses a method for preparing a drug for significantly reducing the number of goblet cells and mast cells on the nasal mucosa and inhibiting the levels of inflammatory factors IL-4, IL-5 and IL-13 in serum.

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