Application of pinoresin-4-O-glucoside in preparation of medicine for treating allergic asthma disease

Abstract

The invention discloses an application of pinoresin-4-O-glucoside in preparation of a medicine for treating allergic asthma diseases, and according to the application provided by the invention, the pinoresin-4-O-glucoside can inhibit expression and activity of induced nitric oxide synthase, remarkably improve airway hyperresponsiveness, relieve lung inflammation and relieve expression of asthma related indexes, and can be used for treating allergic asthma diseases. The application has a prospect of developing a novel anti-asthma medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese medicine research and development, and specifically to the application of pinoresinol-4-O-glucoside in the preparation of medicines for treating allergic asthma. Background Art

[0002] Asthma is a serious health problem that exists all over the world, seriously affecting people's daily lives and sometimes even causing fatal injuries. Allergic asthma is a heterogeneous airway conduction disorder, including chronic airway inflammation, decreased airway function and airway tissue remodeling. Its main pathological features are mucus hypersecretion, airway obstruction, airway inflammation and airway hyperresponsiveness. Asthma is believed to be caused by the interaction of genetic factors and environmental influences, such as the infection dose and infection time of allergens and contact infection. At present, treatment mainly relies on hormone drugs, such as inhaled glucocorticoids, which play a major role in the prevention and treatment of asthma. Its mechanism is to directly inhibit inflammation. Although inhaled glucocorticoids are usually effective, their high doses or long-term use can lead to serious side effects, such as growth retardation, adrenal suppression, fractures, liver damage, cancer and stroke risks, which makes it face a long-term dilemma in clinical use.

[0003] In recent years, TCM has gained attention as a complementary or alternative treatment for diseases. Cistanche deserticola, a perennial herb with a parasitic growth habit, is a precious Chinese medicine that grows mainly in arid regions of the northern hemisphere. Its use in China can be traced back to ancient texts such as Shennong's Materia Medica. Cistanche deserticola is highly regarded for its ability to enhance kidney function, increase vitality, and improve blood quality. Modern scientific research has revealed its multifaceted biological activities, including anti-inflammatory, antioxidant, anti-aging, and immune-enhancing properties. Studies have explored its potential therapeutic applications in diseases such as diabetes, where it has shown good results in regulating postprandial blood glucose levels and improving insulin resistance. In addition, Cistanche deserticola can also reduce damage to the blood-brain barrier after ischemic stroke by improving neuroinflammation. In respiratory diseases, echinacea, a bioactive compound in Cistanche deserticola, is expected to reduce oxidative stress-induced endothelial cell death, thereby alleviating lung injury symptoms, but no researchers have tried it in the treatment of asthma.

[0004] In summary, given the anti-inflammatory and antioxidant effects of Cistanche deserticola, we believe that discovering the efficacy of Cistanche deserticola in asthma and exploring its specific substances and mechanisms of action are of great technical significance for improving and enhancing the prevention and treatment of the disease. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide an application of pinoresin-4-O-glucoside in the preparation of a drug for treating allergic asthma, which can inhibit the expression and activity of inducible nitric oxide synthase, significantly improve airway hyperresponsiveness, reduce lung inflammation, and reduce the expression of asthma-related indicators, and has the prospect of developing new anti-asthma drugs.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The use of pinoresin-4-O-glucoside in the preparation of a drug for treating allergic asthma is characterized in that the chemical structural formula of the pinoresin-4-O-glucoside is as follows:

[0008]

[0009] Cistanche deserticola has a good therapeutic effect on asthma. The inventors conducted a joint analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) technology and the TCMSP database and concluded that the active substance in Cistanche deserticola that has an effect on allergic asthma is pinoresin-4-O-glucoside, and its mechanism is to inhibit the expression and activity of iNOS. Cell and biomolecule-related experiments were used to verify the direct interaction between pinoresin-4-O-glucoside and iNOS.

[0010] The full name of pinoresinol-4-O-BETA-D-pyranoglucoside in Chinese is (+)-Piresil-4-O-beta-D-glucopyraside, and its molecular formula is C 26 H 32 O 11 , CAS number is 69251-96-3.

[0011] Furthermore, the drug is a drug containing pinoresin-4-O-glucoside, and the drug also includes a pharmaceutically acceptable excipient.

[0012] Furthermore, the excipient is one of starch, dextrin, lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, polyethylene glycol, magnesium stearate and micro-powder silica gel, or a mixture of any of them.

[0013] Furthermore, the dosage form of the drug is tablets, granules, capsules, liquid dosage forms, gas dosage forms or pills.

[0014] Furthermore, the tablet is a sustained-release tablet, a controlled-release tablet, a dispersible tablet, an orally disintegrating tablet, a chewable tablet or a buccal tablet, the capsule is a soft capsule, the liquid dosage form is a syrup, an emulsion or an oral solution, and the gas dosage form is an aerosol or a spray.

[0015] Furthermore, the drug is used for at least one of (a) to (c):

[0016] (a) Improve airway hyperresponsiveness and reduce the Penh value, an indicator for quantifying tracheobronchial contractility;

[0017] (b) Reduce lung inflammation, reduce lung neutrophil and eosinophil infiltration, and reduce bronchial mucus secretion;

[0018] (c) Inhibit the expression of asthma-related indicators IgE, IL-5, and IL-13.

[0019] The technical solution of the present invention achieves the following beneficial technical effects:

[0020] 1. Use of pinoresin-4-O-glucoside of the present invention in the preparation of drugs for treating allergic asthma. Pinoresin-4-O-glucoside can significantly improve the pathological changes in the lung tissue of mice with house dust mite-sensitized asthma, inhibit the increase of inflammatory factors, and can be used to prepare drugs for preventing or treating asthma.

[0021] 2. Use of pinoresin-4-O-glucoside of the present invention in the preparation of drugs for treating allergic asthma. Pinoresin-4-O-glucoside is a small molecule compound that can inhibit the expression and activity of inducible nitric oxide synthase (iNOS), significantly improve airway hyperresponsiveness, reduce lung inflammation, and reduce the expression of asthma-related indicators, and has the prospect of developing new anti-asthma drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the method of treating mice with Cistanche deserticola of the present invention and the effect verification;

[0023] A is the process of constructing the mouse asthma model and the Cistanche deserticola treatment model, B is the change of Penh at different concentrations of methacholine, C is the count of various types of cells in the mouse bronchoalveolar lavage fluid, D is the analysis of mouse lung pathological sections, and E is the immunoenzyme-linked analysis of various asthma markers;

[0024] Figure 2 It is a schematic diagram of the liquid chromatography-high resolution mass spectrometry analysis results of the Cistanche deserticola of the present invention and the docking results of pinoresin-4-O-glucoside and iNOS protein molecule;

[0025] A is the total mass spectrometry result of each component of Cistanche deserticola, B is the retention time of pinoresin-4-O-glucoside passing through the chromatographic column, C is the mass-to-charge ratio of pinoresin-4-O-glucoside, and D is the result diagram of the docking of pinoresin-4-O-glucoside and iNOS molecule;

[0026] Figure 3 This is a schematic diagram of the inference verification that the Cistanche deserticola and its active substances of the present invention treat asthma by inhibiting the expression of iNOS;

[0027] A is the qPCR result of iNOS in mouse lung tissue, B is the Western blot result of iNOS after liposin-4-O-glucoside treated non-epithelial cells, C is the immunofluorescence result of iNOS in mouse lung tissue, and D is the experimental result of the effect of liposin-4-O-glucoside on iNOS activity. DETAILED DESCRIPTION

[0028] The principles and features of the present invention are described below in conjunction with specific drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0029] The present invention provides the use of pinoresin-4-O-glucoside in the preparation of a medicament for treating allergic asthma. The chemical structural formula of pinoresin-4-O-glucoside is as follows:

[0030]

[0031] Cistanche deserticola has a good therapeutic effect on asthma. The inventors conducted a joint analysis using liquid chromatography-high resolution mass spectrometry (LC-HRMS) technology and the TCMSP database and concluded that the active substance in Cistanche deserticola that has an effect on allergic asthma is pinoresin-4-O-glucoside, and its mechanism is to inhibit the expression and activity of iNOS. Cell and biomolecule-related experiments were used to verify the direct interaction between pinoresin-4-O-glucoside and iNOS.

[0032] The full name of pinoresinol-4-O-BETA-D-pyranoglucoside in Chinese is (+)-Piresil-4-O-beta-D-glucopyraside, and its molecular formula is C 26 H 32 O 11 , CAS number is 69251-96-3.

[0033] 1. Experimental materials:

[0034] All experimental mice were C57BL / 6 mice, and all animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Southwest Jiaotong University.

[0035] Mle12 mouse alveolar epithelial cells were purchased from Wuhan Punosai Biotechnology Co., Ltd.

[0036] 2. Experimental drugs and reagents

[0037] Cistanche deserticola;

[0038] House dust mites, a product of Greer Labs, USA;

[0039] DMEM high-glucose culture medium, a product of Gibco;

[0040] Fetal bovine serum, a product of Sijiqing Company;

[0041] RNA extraction reagent (Trizol), RNA reverse transcription kit, fluorescent quantitative PCR reagent (2×Taq SYBR GreenqPCR Mix), products of Novezan;

[0042] RIPA lysis buffer, protease phosphatase inhibitors, Triton X-100, 4% paraformaldehyde, sodium dodecylbenzene sulfonate, N,N-methylenebisacrylamide, skim milk powder, etc., products of Solebao Company;

[0043] The membrane is polyvinylidene fluoride (PVDF) membrane, a product of Merck;

[0044] Diffusion staining solution, a product of Biyuntian Company;

[0045] Hematoxylin and eosin staining solution, product of Solebol;

[0046] RIPA protein extract, a product of Bio-Tech;

[0047] Glycogen staining solution, product of Solebol;

[0048] BCA protein concentration determination kit, a product of Solebao Company;

[0049] IgE, IL-5, IL-13 ELISA kits, products of China ELISA Co., Ltd.;

[0050] Nitric oxide enzyme activity assay kit, a product of Beyotime;

[0051] iNOS antibody, a product of Mitaka Corporation;

[0052] Alexa Fluor 555-conjugated goat anti-rabbit IgG H&L, a product of abcam;

[0053] Nuclear dye (DAPI), a product of Bio-Time;

[0054] Hoechst dye, a product of Bio-Tech;

[0055] RNA extraction and qPCR reagents were products of Novezan;

[0056] Pinoresin-4-O-glucoside, CAS No.: 69251-96-3, product of MCE.

[0057] 3. Experimental instruments

[0058] Full body scanner, a product of Tawang Company;

[0059] Micro-spectrophotometer, product of Analytik;

[0060] Real-time fluorescence quantitative PCR instrument, protein electrophoresis instrument, products of Bio-red;

[0061] Fluorescence confocal microscope, product of Olmpus;

[0062] Electronic tablet imaging system, a product of e-blot;

[0063] Example 1: Verification of the efficacy of Cistanche deserticola in treating allergic asthma

[0064] 1. Preparation of Cistanche deserticola

[0065] The Cistanche deserticola was ground into powder, dissolved in purified water at a temperature range of 37-42°C, filtered with a 100 μm filter, and then vacuum-freeze-dried. The obtained powder was weighed and prepared at a concentration of 10 μg / μl.

[0066] 2. Construction of mouse allergic asthma model and Cistanche deserticola treatment model

[0067] After one week of acclimatization, 6-8-week-old C57BL / 6139 mice with similar body weight were selected and randomly divided into a control group (PBS), a Cistanche deserticola group, a house dust mite group, and a treatment group (house dust mite + Cistanche deserticola group).

[0068] according to Figure 1 (A) As shown in the timeline, mice in the house dust mite group and the treatment group were injected with 3 μg HDM by tracheotomy on the first day of the experiment, and 10 μg house dust mites were dripped into the nose every day for 6-10 days of the experiment; both the treatment group and the Cistanche deserticola group were gavaged with 50 mg / kg Cistanche deserticola solution every day during the experiment, and the mice in the house dust mite group and the treatment group were replaced with PBS of the same concentration at the same time. On the 14th day, all mice in the groups were euthanized and the specimens were collected for later use.

[0069] 3. Mouse airway hyperresponsiveness assay

[0070] Airway responsiveness was measured non-invasively using a whole body plethysmograph. Mice were stimulated with different concentrations of aerosolized methacholine (0, 3.125, 6.25, 12.5, 25, and 50 mg / mL). The Penh value was recorded for 10 minutes after each stimulation, and a Penh value was obtained every 10 seconds. For each concentration, the average of the 30 most recent Penh values ​​was taken as the Penh value of the mouse at the current acetylcholine concentration. The Penh value is an indicator for quantifying tracheobronchial contractility and can reflect the high responsiveness of the airways. The larger the value, the higher the airway responsiveness.

[0071] The results are as follows Figure 1 As shown in (B), the curve graph represents the gradual increase in airway reactivity of mice with the increase in the concentration of physicochemical methacholine. When the concentration of methacholine increased to 25 mg / ml, the airway reactivity of mice in the house dust mite group increased significantly, but after treatment with Cistanche deserticola, the airway reactivity was significantly improved, which indicates that the treatment of Cistanche deserticola has a certain inhibitory effect on the airway hyperresponsiveness of allergic asthma.

[0072] 4. Total cell count and cell classification count of mouse bronchoalveolar lavage fluid

[0073] After killing the mice, the lung tissues were taken and 800 μL of PBS was drawn with a syringe to lavage the lungs. The lavage fluid was collected and centrifuged at 800 g for 5 min, then resuspended with 200 μL of PBS and counted. 30 μL of alveolar lavage fluid was evenly spread on the adhesion slide, and then dried and collected for Dif staining, and finally cell classification and counting were performed under a microscope.

[0074] The results are as follows Figure 1 As shown in (C), the total cells, neutrophils and eosinophils in the bronchoalveolar lavage fluid of mice increased significantly after treatment with house dust mites, and decreased after intervention with Cistanche deserticola, indicating that Cistanche deserticola has a significant inhibitory effect on the infiltration of inflammatory cells in the lungs in allergic asthma, which is helpful in alleviating lung inflammation and allergic asthma-related symptoms.

[0075] 5. Pathological analysis of mouse lung tissue

[0076] Fresh lung tissue was fixed with 10% paraformaldehyde for 24 h, dehydrated in alcohol, embedded in paraffin, and then cut into 5 μm thin slices. After staining with hematoxylin and eosin staining solution, the inflammatory cell infiltration in the mouse lungs was evaluated, and after staining with glycogen staining solution, the airway mucus secretion of the mouse was evaluated.

[0077] The results are attached Figure 1 As shown in (D), the degree of inflammatory cell infiltration and mucus secretion around the airways of mice increased after treatment with house dust mites, and decreased after intervention with Cistanche deserticola.

[0078] 6. Enzyme-linked immunosorbent assay (ELISA)

[0079] The total IgE concentration in serum and the interleukin concentration in BALF were quantified using ELISA kits provided by commercial suppliers according to the manufacturer's instructions.

[0080] The results are as follows Figure 1 As shown in (E), various asthma-related indices were increased in the serum and bronchoalveolar lavage fluid of mice treated with house dust mites, and decreased after intervention with Cistanche deserticola.

[0081] In summary, Cistanche deserticola can reduce the Penh value and significantly improve airway hyperresponsiveness; reduce the infiltration of neutrophils and eosinophils in the lungs, as well as bronchial mucus secretion, thereby reducing lung inflammation and inhibiting the expression of asthma-related indicators IgE, IL-5, and IL-13.

[0082] Example 2: Cistanche deserticola reduces the expression of inducible nitric oxide synthase (iNOS) in the lung

[0083] Clinically, the detection of exhaled nitric oxide (FeNO) is an important indicator for evaluating the degree of lung inflammation in asthma. Studies have found that FeNO mainly originates from lung epithelial cells and is produced in large quantities by iNOS. Through the above research, it was found that Cistanche deserticola is of great help in alleviating lung inflammation, so iNOS was used as the main target for subsequent mechanism exploration.

[0084] 1. RNA extraction from mouse lung tissue

[0085] Cut the mouse lung tissue into pieces, put it into a 1.5ml EP tube, add 1ml total RNA extraction reagent and grind it thoroughly with a grinder, let it stand on ice for 5 minutes, add 200μl chloroform, shake and mix until milky white, let it stand on ice for 10 minutes, centrifuge it at 12000g for 15 minutes, draw the upper colorless aqueous phase into a new EP tube and add an equal volume of isopropanol, let it stand on ice for 10 minutes, centrifuge it at 12000g for 10 minutes, and you can see white flake precipitate, which is total RNA. Pour out the isopropanol, add 1ml 75% ethanol and float the white flake precipitate, let it stand on ice for 5 minutes after mixing, centrifuge it at 12000g for 5 minutes, draw all the ethanol, add enzyme-free water to fully dissolve the precipitate after it is slightly dry, measure the concentration and purity of the extracted total RNA with a spectrophotometer, and carry out subsequent experiments. The rest is stored at -80℃.

[0086] Real-time fluorescence quantitative PCR analysis (qPCR): The extracted RNA was reversed into cDNA using a reverse transcription kit, and qPCR was performed using ChamQ SYBR qPCR Master Mix to detect the relative expression of iNOS. Gapdh was used as the internal reference gene during the detection. -△△CtThe relative expression of mRNA was calculated by this method.

[0087] The relevant primers were designed as follows:

[0088] Targeting gapdh:

[0089] Forward: 5'-AGGTCGGTGTGAACGGATTTG-3';

[0090] Reverse: 5'-GGGGTCGTTGATGGCAACA-3';

[0091] Targeting iNOS:

[0092] Forward: 5'-CTCTTCGACGACCCAGAAAC-3';

[0093] Reverse: 5'-CAAGGCCATGAAGTGAGGCTT-3'.

[0094] The results are attached Figure 3 (A) As shown, the expression of iNOS in the lung was increased after house dust mite treatment and decreased after Cistanche deserticola intervention.

[0095] 2. Confocal Microscopy Image Analysis

[0096] After dewaxing, hydration and antigen retrieval, the paraffin sections of lung tissue were permeabilized with PBS solution containing 0.3% Triton X-100 at room temperature for 15 minutes, and then blocked with PBS solution supplemented with 10% goat serum at 37°C for 1 hour. After blocking, they were incubated with iNOS antibody (1:1000) at 4°C overnight. Subsequently, Alexa Fluor 555-labeled goat anti-rabbit IgGH&L conjugate was applied, incubated at 37°C for 1 hour, cell nuclei were stained with DAPI for 5 minutes, and then sealed with a solution containing 50% glycerol, and the specimens were observed using a fluorescence confocal microscope and analyzed using CellSens application kit software.

[0097] The results are as follows Figure 3 As shown in (C), the red fluorescence intensity in the figure reflects the expression level of iNOS in different parts. It can be seen that the expression level of iNOS in the airway epithelium was significantly increased in mice treated with house dust mites, and decreased after intervention with Cistanche deserticola.

[0098] The above experimental results show that the main target of Cistanche deserticola in reducing allergic asthma symptoms and lung inflammation is iNOS.

[0099] Example 3: Analysis of active ingredients in Cistanche deserticola

[0100] Liquid chromatography-high resolution mass spectrometry was used to qualitatively analyze Cistanche deserticola using LC-HRMS (Nexera UHPLC LC-30A and ABSCIEX QtofX500R) combined with Turbo V TM Source and SCIEX OS software (version 1.7) were used to identify the main active ingredients in Cistanche deserticola for the treatment of asthma by comparing the retention time and mass spectra with samples and standards.

[0101] The samples were separated using an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1×100 mm) at a column temperature of 40°C and a flow rate of 0.4 mL / min.

[0102] The mobile phase was 0.1% formic acid (A) and acetonitrile (B), and the gradient was: 2% B, 1 min; 98% B soak for 15 min; 98% B soak for 20 min; 2% B, 20.5 min; 2% B, injection for 25 min, injection volume 1.0 μm, injection temperature 15°C.

[0103] Mass spectrometry analysis was performed using an electrospray ionization (ESI) source under the information dependent acquisition (IDA) model. The positive source parameters were: ion source gas 1: 50 psi; ion source gas 2: 50 psi; curtain gas: 35 psi; CAD gas: 7; temperature: 450°C; and spray voltage: 5500V.

[0104] The mass range of TOF-MS is 100-1000Da; focus potential (DP): 80V; DP diffusion: 0; collision energy (CE): 10V; CE diffusion: 0; accumulation time: 0.1s; IDA standard: small molecules.

[0105] The mass range of TOF-MS / MS is 50-1000Da; Dp: 80v; DP diffusion: 0; Ce: 40v; CE diffusion: 20v; accumulation time: 0.05s.

[0106] Table 1 shows the top 30 active ingredients of Cistanche deserticola in terms of abundance analyzed by liquid chromatography-high resolution mass spectrometry; Table 2 shows 9 active ingredients of Cistanche deserticola screened from the TSCMP database based on bioavailability (OB) (≥20%) and drug similarity (DL) (≥0.2) standards.

[0107] Table 1 Top 30 components in Cistanche ranked by abundance

[0108]

[0109]

[0110] Table 2 Nine active components of Cistanche were screened from the TSCMPdatabase(OB≥20%,DL≥0.2)

[0111]

[0112] Combination Figure 2 (A) The top 30 rankings of abundance in Table 1 and the main active ingredients screened in Table 2, pinoresin-4-O-glucoside as the only repeated active substance, is the main substance that plays a role in the treatment of asthma by Cistanche deserticola. Its mass spectrometry analysis results are shown in the attached Figure 2 As shown in (BC), the mass-to-charge ratio is 521.2 and the retention time is 5.3 minutes.

[0113] Example 4: Pinoresin-4-O-glucoside inhibits the expression and activity of nitric oxide synthase (iNOS)

[0114] 1. Molecular docking: The crystal structure of iNOS was retrieved from the RCSB protein database with the code PDB ID: 1DWV, and the three-dimensional structure of pinoresin-4-O-glucoside was obtained from the PubChem database. Protein and ligand structures were prepared in pdbqt file format using AutoDock Tools 1.5.7. Molecular docking studies were performed using AutoDock Vina 1.1.2. The protein residues involved in ligand binding during docking were analyzed and visualized using the PLIP web server and PyMol software.

[0115] The results are as follows Figure 2 As shown in (D), pinoresin-4-O-glucoside has good binding with iNOS amino acids such as arginine, glutamic acid, and tryptophan.

[0116] 2. Cell culture: Mouse lung epithelial cells mle12 were selected as research cells and cultured in DMEN / F-12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution in a culture environment of 37°C and 5% CO2.

[0117] 3. Drug treatment: Dissolve pinoresin-4-O-glucoside in dimethyl sulfoxide for later use. After the cells grow to the logarithmic growth phase, the cells are divided into 4 groups, namely, the control group, the drug control group, the house dust mite group, and the house dust mite plus drug group.

[0118] On the first day of the experiment, 30 μM of pinoresin-4-O-glucoside was added to the drug group and the drug control group, and an equal amount of dimethyl sulfoxide was added to the control group. On the third day of the experiment, 30 μg / ml of house dust mites were added to the house dust mite group and the house dust mite plus drug group, respectively. On the fourth day of the experiment, cells were collected for subsequent experiments.

[0119] 4. Western blot detection: Cellular proteins were extracted with RIPA buffer, and the protein concentration was determined using a BCA protein assay kit. Equal amounts of total protein in each lane were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane. The membrane was then incubated with a rabbit anti-mouse iNOS antibody (Abcam, 1:1000 dilution) at 4°C overnight, then exposed to a horseradish peroxidase-conjugated secondary antibody for 1 h, and the protein was subsequently detected using an electronic compression imaging system.

[0120] The results are attached Figure 3 As shown in (B), the darkness of the band represents the expression level of iNOS. The expression of iNOS in epithelial cells treated with house dust mites increased, and it decreased significantly after treatment with pinoresin-4-O-glucoside.

[0121] 5. iNOS enzyme activity detection: Using the above cell culture and drug treatment methods, culture cells in a confocal dedicated glass bottom culture dish, stain with a nitric oxide enzyme (iNOS) activity detection kit, stain the cell nucleus with Hoechst dye, observe under a confocal microscope, and analyze using the CellSens application kit software.

[0122] The results are attached Figure 3 As shown in (D), the green fluorescence intensity represents the activity of iNOS. The higher the iNOS activity, the stronger the green fluorescence. It can be seen that the iNOS activity increased after treatment with house dust mites, but decreased significantly after treatment with pinoresin-4-O-glucoside, which indicates that pinoresin-4-O-glucoside has the effect of inhibiting iNOS activity.

[0123] From the above, we can see that the target of the active substance pinoresin-4-O-glucoside in Cistanche deserticola for the treatment of allergic asthma is iNOS. Pinoresin-4-O-glucoside has an inhibitory effect on the expression of iNOS in lung epithelial cells, and enzyme activity experiments have proved that pinoresin-4-O-glucoside has an inhibitory effect on the activity of iNOS. Molecular docking experiments have proved that pinoresin-4-O-glucoside has a direct interaction with iNOS.

[0124] Therefore, Cistanche deserticola mainly plays a role in the treatment of allergic asthma through its active substance, pinoresin-4-O-glucoside, and its mechanism is to inhibit the expression and activity of inducible nitric oxide synthase (iNOS), significantly improve airway hyperresponsiveness, reduce lung inflammation, and reduce the expression of asthma-related indicators.

[0125] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. Use of pinoresinol-4-O-glucoside in the preparation of a drug for treating allergic asthma, characterized in that: The chemical structural formula of the pinoresin-4-O-glucoside is as follows:

2. The use according to claim 1, characterized in that: The medicine contains pinoresin-4-O-glucoside and further comprises a pharmaceutically acceptable excipient.

3. The use according to claim 2, characterized in that: The excipient is one of starch, dextrin, lactose, microcrystalline cellulose, hydroxypropyl methylcellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, polyethylene glycol, magnesium stearate and micro-powder silica gel, or a mixture of any of the above.

4. The use according to claim 2, characterized in that: The dosage form of the medicine is tablet, granule, capsule, liquid dosage form, gas dosage form or dripping pill.

5. The use according to claim 4, characterized in that: The tablet is a sustained-release tablet, a controlled-release tablet, a dispersible tablet, an orally disintegrating tablet, a chewable tablet or a buccal tablet; the capsule is a soft capsule; the liquid dosage form is a syrup, an emulsion or an oral solution; and the gas dosage form is an aerosol or a spray.

6. The use according to claim 1, characterized in that: The drug is used for at least one of (a) to (c): (a) Improve airway hyperresponsiveness and reduce the Penh value, an indicator for quantifying tracheobronchial contractility; (b) Reduce lung inflammation, reduce lung neutrophil and eosinophil infiltration, and reduce bronchial mucus secretion; (c) Inhibit the expression of asthma-related indicators IgE, IL-5, and IL-13.

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