Traditional Chinese medicine delivery system of temperature-sensitive gel for allergic rhinitis based on cocklebur fruit formula
By constructing a Xanthium sibiricum-based thermosensitive gel drug delivery system and optimizing the formula using network pharmacology and response surface methodology, a thermosensitive gel with clearly defined components was prepared. This solved the problems of quality fluctuations and side effects in the treatment of allergic rhinitis and achieved a highly efficient and stable nasal drug delivery effect.
Patent Information
- Application Number
- CN202511565543.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-30
- Publication Date
- 2025-11-28
AI Technical Summary
Existing treatments for allergic rhinitis have side effects such as nasal dryness and chronic rhinitis that can occur with long-term use. Furthermore, the quality of traditional Chinese medicine preparations fluctuates greatly, and there is a lack of stable and rapid preparation methods.
A thermosensitive gel drug delivery system based on Xanthium sibiricum was constructed. The formulation was optimized by network pharmacology prediction and response surface methodology. Combined with a thermosensitive gel matrix, a traditional Chinese medicine drug delivery system with clear components and excellent efficacy was prepared. The thermosensitive gel was used to achieve long-term retention by instantaneous gelation at nasal cavity temperature. Combined with a mucosal adhesive, sustained release was achieved.
It significantly improves the residence time and local concentration of drugs in the nasal cavity, enhances bioavailability and medication adherence, and has better efficacy than traditional methods, while maintaining controllable quality.
Smart Images
Figure CN121015553A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of pharmaceutical preparations, and relates to a traditional Chinese medicine drug delivery system, in particular to a traditional Chinese medicine drug delivery system of a temperature-sensitive gel for allergic rhinitis based on a Xiang'ezi formula. BACKGROUND
[0002] Allergic rhinitis (AR) is a global high-incidence disease, and its core pathogenesis involves immunoglobulin E-mediated type I hypersensitivity. Clinically, it is often manifested as four typical symptoms of paroxysmal sneezing, clear watery nasal discharge, nasal itching and nasal congestion, and can be accompanied by hyposmia, sleep disorders and the like when severe, which significantly reduces the quality of life of patients. At present, the first-line drugs in clinical treatment are mainly antihistamines and nasal glucocorticoids, but long-term use can easily cause nasal dryness, and even cause acute and chronic sinusitis, chronic rhinitis and post-nasal drip syndrome, and it is urgent to develop a new type of treatment strategy which is safer and more effective.
[0003] Network pharmacology research reveals that TCM clinical treatment of rhinitis has the advantages of multiple targets, multiple pathways and reversibility. The application of the overall concept and the core idea of syndrome differentiation and treatment in TCM theory in the treatment of rhinitis can not only inhibit local allergic reactions and control symptoms at various stages of rhinitis, but also effectively restore immune balance and prevent recurrence, and has a significant clinical effect on the treatment of allergic rhinitis.
[0004] Taking the Xiang'ezi compound as the object, a "component-target-gel" trinity drug delivery system is constructed, the AGE-RAGE pathway predicted by network pharmacology is integrated, the prescription process of the temperature-sensitive gel is optimized by the response surface method, and molecular docking and multi-dimensional pharmacodynamic evaluation are combined, so as to break through the bottleneck of traditional Chinese medicine preparations in terms of extensive and low efficiency.
[0005] The multi-component synergistic effect makes the compound not only able to relieve symptoms such as nasal congestion and runny nose, but also able to intervene in the pathological process of rhinitis from multiple pathways such as immune regulation, anti-inflammatory and antibacterial, and has a comprehensive treatment advantage over single antihistamine drugs or traditional Xiang'ezi powder for AR.
[0006] Based on this, a traditional Chinese medicine drug delivery system of a temperature-sensitive gel for allergic rhinitis based on a Xiang'ezi formula is proposed. SUMMARY
[0007] The purpose of the present application is to solve the above-mentioned problems existing in the prior art, and a traditional Chinese medicine drug delivery system of a temperature-sensitive gel for allergic rhinitis based on a Xiang'ezi formula is proposed. The technical problem to be solved by the present application is how to realize stable and rapid preparation of a temperature-sensitive gel for allergic rhinitis, which is quality controllable, mechanism clear and curative effect excellent.
[0008] The purpose of the present application can be realized by the following technical scheme: A preparation method of a traditional Chinese medicine delivery system of a temperature-sensitive gel for allergic rhinitis based on Xiang'erzi Fang, comprising the following preparation steps: S1. Initial formulation of Xiang'erzi San for treating rhinitis: optimize the existing formula, i.e. Formula One, which comprises the following components in parts by weight: Xiang'erzi 9, Bai Zhi 8, Xin Yi 10, and Bo He 5, to Formula Two according to clinical experience of traditional Chinese medicine, which comprises the following components in parts by weight: Xiang'erzi 9, Bai Zhi 8, Xin Yi 10, Bo He 5, Fang Feng 5, and Huang Qi 5; S2. Network pharmacology prediction of raw materials: predict the core target points and pathways of Formula Two for treating allergic rhinitis using network pharmacology, and further optimize the extraction process and ratio of Formula Two by response surface method to obtain Formula Three, which comprises the following components in parts by weight: Xiang'erzi 9, Bai Zhi 8, Xin Yi 10, Bo He 5, Fang Feng 5, Huang Qi 8, Huang Qi 5, and Gan Cao 6; S3. Trial production of raw material level temperature-sensitive gel products: based on Formula One, Formula Two, and Formula Three, respectively, A1. prepare a temperature-sensitive gel matrix; A2. add the mixed materials of Formula One, Formula Two, and Formula Three, respectively, to obtain several corresponding raw material level temperature-sensitive gel solutions; Formula One and Formula Two are control examples to prove that the efficacy of Formula Three is better than that of any one of Formula One and Formula Two, thereby strengthening the demonstration of inventiveness; S4. Raw material level experimental testing: multi-level test evaluation of the appearance, physical stability, and rheological properties of several raw material level temperature-sensitive gel solutions; in vitro experiments and animal model verification of the effectiveness of treating rhinitis; confirmation of the most effective raw material level traditional Chinese medicine formula for treating rhinitis, which is recorded as the basic optimized formula; S5. Network pharmacology prediction of single components: replace the traditional Chinese medicine raw materials in the basic optimized formula with effective component monomers determined by network pharmacology and response surface method, and form several new formulas composed of different types of effective component monomers, which are recorded as primary monomer combination formulas M1, M1, M3, etc.; S6. Preliminary trial production of single component level temperature-sensitive gel products: based on several primary monomer combination formulas M, respectively, A1. prepare a temperature-sensitive gel matrix; B2. add the mixed materials of several primary monomer combination formulas M, respectively, to obtain several corresponding single component level preliminary temperature-sensitive gel solutions; S7. Single component level preliminary experimental testing: multi-level test evaluation of the appearance, physical stability, and rheological properties of several single component level preliminary temperature-sensitive gel solutions; in vitro experiments and animal model verification of the effectiveness of treating rhinitis; confirmation of the most effective single component level traditional Chinese medicine formula for treating rhinitis, which is recorded as Formula Four; S8. Formula upgrading: adjust and optimize the component proportions of each effective component of Formula Four to obtain several new formulas, which are recorded as optimized proportion combination formulas P1, P2, P3, etc.; S9. Trial production of monomer-grade thermosensitive gel products: Trial production of thermosensitive gel products based on optimized ratio combination formula P: A1. Prepare thermosensitive gel matrix; C2. Add several mixtures of optimized ratio combination formula P respectively to obtain several corresponding monomer-grade thermosensitive gel solutions. S10. Monomer-level re-experimental testing: Multi-level experimental evaluation of the appearance, physical stability and rheological properties of several monomer-level re-thermosensitive gel solutions; in vitro experiments and animal model verification of the effectiveness in treating rhinitis; confirmation of the most effective traditional Chinese medicine formula for treating rhinitis, which is designated as Formula 5. Formula 5 is the final monomer formula, and the thermosensitive gel prepared based on it has the best properties and therapeutic effect.
[0009] The formulation and preparation method of the thermosensitive gel matrix prepared by A1 are as follows: poloxamer 188 (1%, w / v), poloxamer 407 (20%, w / v), polyethylene glycol 6000 (1%, w / v), HP-β-CD (3%, w / v), methylcellulose (2%, w / v) and chitosan quaternary ammonium salt (1%, w / v), dissolved and evenly dispersed in an appropriate amount of purified water to form the thermosensitive gel matrix.
[0010] The method of adding A2, B2 and C2 to the mixture of each formulation is as follows: the mixture of each formulation and purified water are added to the thermosensitive gel matrix while stirring to obtain a mixed solution. The composition ratio of the mixture of each formulation is 72%, w / v.
[0011] The method for preparing the temperature-sensitive gel solution from the mixed solution is as follows: the mixed solution is refrigerated at 4°C for more than 24 hours to swell, then restored to room temperature, and purified water is added to the prescribed amount to obtain the temperature-sensitive gel solution. The prescribed amount of purified water is the solution volume in terms of mass-volume concentration.
[0012] The pH of the thermosensitive gel solution prepared by Formula 5 is 5.6 ± 0.1.
[0013] The thermosensitive gel solution prepared by Formula 5 has the following appearance: at 25°C, it is a homogeneous, free-flowing sol; when the temperature is raised to 32.6°C, a significant thermo-induced reversible phase transition occurs, forming a gel with elasticity and strong adhesion, indicating that its appearance meets the requirements.
[0014] The physical stability of the thermosensitive gel solution prepared by Formula 5 was tested by centrifugation at 3000 r / min for 30 min. No layering, precipitation or phase separation was observed, indicating that it has good physical stability.
[0015] The rheological property test of the temperature-sensitive gel of the formula five is carried out by using an Anton Paar Physica MCR102e rheometer, taking an appropriate amount of Xanthium compound temperature-sensitive gel and placing it on a PP25 stainless steel parallel plate with a diameter of 25 mm, setting the test gap parameter to 0.6 mm, including gel linear viscoelastic region test, temperature sweep test, frequency sweep test and 3ITT test.
[0016] The T test procedure includes three stages: the first stage, 0.1% strain for 120s, low shear simulating the standing state; the second stage, 100% strain for 40s, high shear simulating the drug administration shear; the third stage, 0.1% strain for 300s, low shear observing the structure recovery; the viscosity data is collected at 10 points / s for each stage.
[0017] A temperature-sensitive gel drug delivery system for treating allergic rhinitis prepared by the above preparation method.
[0018] The application of a temperature-sensitive gel drug delivery system in the preparation of a medicine for treating allergic rhinitis, the temperature-sensitive gel in a free-flowing sol state at room temperature of 25°C is placed in the nasal cavity, the respiratory airflow causes the temperature of the temperature-sensitive gel to rise to 32.6°C, a significant thermoreversible phase transition occurs, forming a gel state with certain elasticity and strong adhesion. This transition can effectively resist ciliary clearance, significantly prolong the residence time of the drug at the action site, and achieve sustained release.
[0019] Compared with the prior art, the temperature-sensitive gel for allergic rhinitis based on the Xanthium formula has the following advantages: A closed-loop iterative R&D system of "clinical experience-network pharmacology prediction-response surface method process optimization-multilevel pharmacodynamics verification" is constructed. Starting from the raw material formula, the basic optimized formula is screened through rigorous comparative tests, and then the medicinal materials are innovatively replaced by effective ingredient monomers, and after two rounds of upgrading of "monomer combination screening" and "proportion optimization", the optimal monomer formula with clear ingredients and accurate proportion is finally obtained.
[0020] The temperature-sensitive gel matrix formula adopted is not a conventional choice, but is specially designed for nasal administration, which can realize the dual functions of flowing at room temperature for easy administration and instantaneous gelation at the temperature of the nasal cavity for long-term retention. Combined with mucosal adhesives, the local concentration and action time of the drug are greatly improved. The bioavailability of the drug and the medication compliance of the patient are significantly improved.
[0021] The method can prepare a mass controllable, mechanism clear and excellent curative effect traditional Chinese medicine product. The final product is composed of chemical monomers, completely solves the pain point of large quality fluctuation of traditional Chinese medicine raw materials, realizes the standardization and quality controllability of the product. And through comprehensive in vitro and in vivo experiments, it is confirmed that the curative effect is not only significantly better than the previous formula and positive control drug, and the mechanism is verified at the molecular biology level. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 is a preparation process schematic diagram of the present application.
[0023] Figure 2 is a preparation detail schematic diagram of the present application.
[0024] Figure 3 is a technical route schematic diagram of the present application.
[0025] Figure 4 is a linear viscoelastic region test result schematic diagram of the gel of the present application Figure 1 .
[0026] Figure 5 is a linear viscoelastic region test result schematic diagram of the gel of the present application Figure 2 .
[0027] Figure 6 is a G' and G'' curve diagram of the temperature-sensitive gel of the present application with temperature change.
[0028] Figure 7 is a frequency scanning result schematic diagram of the temperature-sensitive gel of the present application.
[0029] Figure 8 is a thixotropy test result of the temperature-sensitive gel of the present application.
[0030] Figure 9 is a Venn diagram of AR prediction target of the present application.
[0031] Figure 10 is a Venn diagram of CEZY and AR target of the present application.
[0032] Figure 11 is a network diagram of CEZY anti-AR "disease-drug-component-intersection target" of the present application.
[0033] Figure 12 is a number table of 10 key compounds of CEZY anti-AR of the present application.
[0034] Figure 13 is a PPI network diagram of "drug-disease" intersection target of the present application.
[0035] Figure 14CEZY anti-AR target PPI network screening diagram of the present application.
[0036] Figure 15 CEZY anti-AR target GO biological process enrichment analysis column chart of the present application.
[0037] Figure 16 CEZY anti-AR target KEGG pathway enrichment analysis bubble chart of the present application.
[0038] Figure 17 CEZY anti-AR 10-position "core target-signal pathway" network diagram of the present application.
[0039] Figure 18 Raw material grading formula table of the present application.
[0040] Figure 19 Monomer table of the main effective component of the present application.
[0041] Figure 20 Monomer grading formula table of the present application.
[0042] Figure 21 Appearance diagram of the temperature-sensitive gel of the present application at 25℃.
[0043] Figure 22 Appearance diagram of the temperature-sensitive gel of the present application at 32.6℃.
[0044] Figure 23 In-vitro experiment mean data table of the present application.
[0045] Figure 24 Animal model verification mean data table of the present application. DETAILED DESCRIPTION
[0046] The following are specific embodiments of the present application and further describe the technical solutions of the present application in conjunction with the accompanying drawings, but the present application is not limited to these embodiments.
[0047] As shown in Figures 1-3 , the preparation method of the traditional Chinese medicine delivery system of the temperature-sensitive gel for allergic rhinitis (AR) based on the Xianfuzi prescription, comprises the following preparation steps: S1. Xianfuzi powder traditional Chinese medicine formula for treating rhinitis is initially proposed: according to the existing formula, i.e. formula one, is optimized, formula one includes the following components in weight parts: Xianfuzi 9, Baizhi 8, Xinyi 10 and Bohe 5, and is optimized to formula two according to the clinical experience of traditional Chinese medicine, formula two includes the following components in weight parts: Xianfuzi 9, Baizhi 8, Xinyi 10, Bohe 5, Fangfeng 5 and Huangqi 5; S2. Raw material level network pharmacology prediction: Use network pharmacology to predict the core target points and pathways of formula two for treating allergic rhinitis, and further optimize the extraction process and ratio of formula two by response surface method to obtain formula three, which comprises the following components in parts by weight: xanthium 9, angelica 8, lonicera 10, mentha 5, sileris 5, scutellaria 8, astragalus 5, and licorice 6; S3. Raw material level temperature-sensitive gel product trial: Based on formula one, formula two and formula three, temperature-sensitive gel products are prepared, A1. Prepare the temperature-sensitive gel matrix; A2. Add the mixture of formula one, formula two and formula three respectively, and obtain several corresponding raw material level temperature-sensitive gel solutions; Formula one and formula two are control examples, which prove that the efficacy of formula three is better than that of any one of formula one and two, thereby strengthening the demonstration of inventiveness; S4. Raw material level experimental test: Multi-level test evaluation, appearance, physical stability and rheological properties of several raw material level temperature-sensitive gel solutions; in vitro experiments and animal model verification, treatment effectiveness of rhinitis; confirm the most effective treatment of rhinitis of the raw material level traditional Chinese medicine formula, which is recorded as the basic optimized formula; S5. Single level network pharmacology prediction: Replace the traditional Chinese medicine raw materials in the basic optimized formula with the effective component monomers determined by network pharmacology and response surface method, and form several new formulas composed of different types of effective component monomers, recorded as primary monomer combination formulas M1, M1, M3...; S6. Single level temperature-sensitive gel product preliminary trial: Based on several primary monomer combination formulas M, temperature-sensitive gel products are prepared, A1. Prepare the temperature-sensitive gel matrix; B2. Add the mixture of several primary monomer combination formulas M respectively, and obtain several corresponding single level preliminary temperature-sensitive gel solutions; S7. Single level preliminary experimental test: Multi-level test evaluation, appearance, physical stability and rheological properties of several single level preliminary temperature-sensitive gel solutions; in vitro experiments and animal model verification, treatment effectiveness of rhinitis; confirm the most effective treatment of rhinitis of the single level traditional Chinese medicine formula, which is recorded as formula four; (as shown in Figure 19 ); S8. Formula upgrade: Adjust and optimize the component ratio of each effective component of traditional Chinese medicine formula four to obtain several new formulas, recorded as optimized ratio combination formulas P1, P2, P3...; S9. Single level temperature-sensitive gel product re-trial: Based on the temperature-sensitive gel product trial of the optimized ratio combination formula P: A1. Prepare the temperature-sensitive gel matrix; C2. Add the mixture of several optimized ratio combination formulas P respectively, and obtain several corresponding single level re-temperature-sensitive gel solutions; S10. Monomer level retest: multi-level test evaluation, the appearance, physical stability and rheological properties of several monomer level retest temperature-sensitive gel solutions; in vitro experiments and animal model validation, effectiveness of treating rhinitis; confirm the most effective traditional Chinese medicine formula for treating rhinitis, which is recorded as Formula Five (as shown in Figure 20 , which is the final monomer formula, and the temperature-sensitive gel prepared based thereon has the best properties and treatment effect.
[0048] The prescription amount composition ratio and preparation method of the temperature-sensitive gel matrix are as follows: poloxamer 188 (1%, w / v), poloxamer 407 (20%, w / v), polyethylene glycol 6000 (1%, w / v), HP-β-CD (3%, w / v), methyl cellulose (2%, w / v), and chitosan quaternary ammonium salt (1%, w / v) are dissolved and uniformly dispersed in an appropriate amount of purified water to obtain the temperature-sensitive gel matrix.
[0049] The method of adding the mixed materials of each formula to the temperature-sensitive gel matrix is to add the mixed materials of each formula and purified water to the temperature-sensitive gel matrix while stirring to obtain a mixed solution, and the composition ratio of the mixed materials of each formula is 72%, w / v.
[0050] The preparation method of the mixed solution to prepare the temperature-sensitive gel solution is to store the mixed solution at 4°C for swelling for more than 24 h, and then restore it to room temperature, and then supplement purified water to the prescription amount, i.e., the volume of the solution with a mass-volume concentration, to obtain the temperature-sensitive gel solution.
[0051] The pH of the temperature-sensitive gel solution prepared by Formula Five is 5.6±0.1.
[0052] The appearance of the temperature-sensitive gel solution prepared by Formula Five is a uniform, free-flowing sol state at 25°C; when the temperature is raised to 32.6°C, a significant thermoreversible phase transition occurs, forming a gel state with elasticity and strong adhesion, indicating that its appearance meets the requirements.
[0053] The physical stability test method of the temperature-sensitive gel solution prepared by Formula Five is to observe no stratification, precipitation or phase separation after centrifugation at 3000 r / min for 30 min, indicating that it has good physical stability.
[0054] The rheological property test method of the temperature-sensitive gel of Formula Five is to use an Anton Paar Physica MCR102e rheometer, take an appropriate amount of xanthium compound temperature-sensitive gel and place it on a PP25 stainless steel parallel plate with a diameter of 25 mm, set the test gap parameter to 0.6 mm, including gel linear viscoelastic region test, temperature sweep test, frequency sweep test and 3ITT test.
[0055] The T-test procedure contains three stages: the first stage, 0.1% strain for 120s, low shear simulating the resting state; the second stage, 100% strain for 40s, high shear simulating the drug administration shear; the third stage, 0.1% strain for 300s, low shear observing the structure recovery; the viscosity data is collected at 10 points / s for each stage.
[0056] A thermosensitive gel drug delivery system for treating allergic rhinitis prepared by the above preparation method.
[0057] The application of a thermosensitive gel drug delivery system in the preparation of a drug for treating allergic rhinitis, the thermosensitive gel in a free-flowing sol state at room temperature of 25°C is placed inside the nasal cavity, the respiratory airflow causes the temperature of the thermosensitive gel to rise to 32.6°C, a significant thermoreversible phase transition occurs, forming a gel state with certain elasticity and strong adhesion, this transition can effectively resist ciliary clearance, significantly prolong the residence time of the drug at the action site, and achieve sustained release.
[0058] Example 1 The formula three includes the following components by weight fraction: Semen Xanthii 9, Radix Angelicae Pubescentis 8, Magnoliae Flos 10, Herba Menthae 5, Saposhnikovia Divaricata 5, Radix Scutellariae 8, Radix Astragali 5, and Glycyrrhiza 6; the above preparation method is used to prepare the raw material grade thermosensitive gel solution three. (As shown in Figure 18 ) Example 2 (M) The formula M is combined with several primary monomers, and the above preparation method is used to prepare the raw material grade thermosensitive gel solutions M1, M1, M3...
[0059] Example 3 (P) The formula P is combined based on the optimized proportion, and the above preparation method is used to prepare the raw material grade thermosensitive gel solutions P1, P2, P3...
[0060] Comparative Example 1 The formula one includes the following components by weight fraction: Semen Xanthii 9, Radix Angelicae Pubescentis 8, Magnoliae Flos 10, and Herba Menthae 5; the above preparation method is used to prepare the raw material grade thermosensitive gel solution one. (As shown in Figure 18 ) Comparative Example 2 The formula two includes the following components by weight fraction: Semen Xanthii 9, Radix Angelicae Pubescentis 8, Magnoliae Flos 10, Herba Menthae 5, Saposhnikovia Divaricata 5, and Radix Astragali 5; the above preparation method is used to prepare the raw material grade thermosensitive gel solution two. (As shown in Figure 18 ) I. Physical stability and rheological property test Rheological property of thermosensitive gel: The physiological temperature of human nasal cavity is about 32-35℃, which meets the pH requirement of 4.5-6.5 for nasal administration of the preparation. Using an Anton Paar Physica MCR102e rheometer, an appropriate amount of Xanthium compound temperature-sensitive gel was placed on a PP25 stainless steel parallel plate with a diameter of 25 mm, and the test gap parameter was set to 0.6 mm.
[0061] Linear viscoelastic region of gel: The relative size of the test results of the linear viscoelastic region of the gel can reflect the integrity and stability of the gel structure in the sample. As shown in Figure 4 and Figure 5 , when the shear strain is greater than 0.2%, G' begins to decrease, and when G'=G'', the gel structure is destroyed and the stability decreases. Therefore, the strain in all dynamic oscillation experiments should be controlled within 0.2%.
[0062] Temperature scanning: The phase transition temperature of the temperature-sensitive gel was determined by oscillation mode, and the temperature was increased at a rate of 2℃ / min from 5-60℃. As shown in Figure 6 , at a lower temperature, the G'' of the temperature-sensitive gel is greater than G', indicating that the temperature-sensitive gel has fluid properties and can flow; when the temperature reaches 28.1℃, G'' intersects with G', and the temperature at this time is the phase transition temperature at which the temperature-sensitive gel begins to change from sol to gel. As the temperature continues to rise, G' is always greater than G'', indicating that it is always in the process of changing to a gel, and the elastic characteristics of the gel dominate; when the temperature rises to 41.1℃, the change of G' and G'' begins to be gentle, indicating that the viscoelasticity tends to be stable, and has changed to a semi-solid gel state with mainly elastic characteristics.
[0063] Frequency scanning: Under oscillation mode, the parameter γ was set to 1%, and the angular frequency scanning range was 0.1-600 rad / s, and the results are shown in Figure 7 . Within 0.1-600 rad / s, the G' curve is always higher than the G" curve, showing obvious elastic properties, and there is no significant frequency dependence, indicating that the internal structure of the temperature-sensitive gel is stable.
[0064] The 3ITT test procedure includes three stages: the first stage (0.1% strain for 120 s, low shear simulating static state), the second stage (100% strain for 40 s, high shear simulating drug administration shear), and the third stage (0.1% strain for 300 s, low shear observing structure recovery), and the viscosity data was collected at a rate of 10 points per second for each stage. As shown in Figure 8 , the recovery rate was calculated to be 77.4% (4566 / 5903x100%), which is lower than the required recovery rate of 80% for nasal administration of the gel, indicating that the currently prepared Xanthium compound temperature-sensitive gel cannot quickly rebuild the gel network after shear stops.
[0065] II. Network pharmacology prediction method 2.1.CEZY active ingredient collection and target point prediction: The relevant chemical components of 8 traditional Chinese medicines in SDQJW, including Bai Zhi, Xinyi, Bohe, Fangfeng, Chao Cang'erzi, Huangqipian, Huangqi, and Gancaopian, were retrieved through the computational systems biology laboratory (TCMSP) (http: / / lsp.nwu.edu.cn / tcmsp.php). The drug-like property (DL) was set to be greater than or equal to 0.18, and the oral bioavailability (OB) was set to be greater than or equal to 30%. The potential effective active ingredients of the 8 traditional Chinese medicines in the prescription were screened, and the related action targets of the active ingredients were retrieved. Then, the official standardized name was obtained by querying the gene abbreviation of each target point through the Uniprot database (https: / / www.uniprot.org / ) and correcting it.
[0066] Since the TCMSP database does not contain information about Chao Cang'erzi, the active ingredients of Chao Cang'erzi were retrieved through literature. The SMILES number of the active ingredients was retrieved through the Pubchem database and input into the Swiss ADME platform for screening. The score of gastrointestinal absorption (GI absorption) was set to be "high", and the drug-like property (Druglike-ness) was at least screened through 2 "Yes". The SMILES number of the screened compounds was input into Swiss Target Prediction to predict the action targets of each chemical component, and the potential action targets of the active ingredients of Chao Cang'erzi were obtained.
[0067] 2.2. AR disease target collection: The search term was set to be "Allergic rhinitis", and the AR disease-related target genes were obtained from the Genecards database (https: / / www.GeneCards.org / ), the human Mendelian genetic synthesis database OMIM (https: / / omim.org / ), and the TTD database (https: / / db.idrblab.net / ttd / ). The first-line western medicine action targets for treating AR disease were supplemented by searching the Drugbank database (https: / / go.drugbank.com / ). After removing the repeated and false positive targets, the AR disease target data were obtained after sorting and analyzing.
[0068] 2.3. Prediction of CEZY anti-AR disease target points and construction of "drug-disease" protein interaction network: In order to clarify the interaction between the target points of the active ingredients of CEZY and the target points of AR disease, the VENNY software was used to take the intersection of the two target points and draw a Venny diagram to find out the mapping. The corresponding intersection target points were obtained and imported into the String database (https: / / www.string-db.org / ), the biological species was set to "Homo sapiens", the minimum interaction threshold was set to "highest confidence" (≫0.9), and the rest were set to default settings. The PPI protein interaction network was obtained and the tsv file was exported, which was input into Cytoscape3.7.2 software. The topological parameter results were calculated, the related data analysis network was constructed, and the core target genes were screened out.
[0069] 2.4. Visualization network construction of "drug-ingredient-target-disease" of CEZY anti-AR: The active ingredients of 8 traditional Chinese medicines in "Xiang'erzi drink" formula and related target genes, AR disease target genes, and "drug-disease" intersection target points were analyzed by Cytoscape3.7.2 software, and the "drug-ingredient-target-disease" visualization network was constructed.
[0070] 2.5. GO and KEGG pathway enrichment analysis: The "drug-disease" intersection target points were imported into the DAVID database (https: / / david.ncifcrf.gov / conversion.jsp) for gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The biological process (BP), molecular function (MF), and cell component (CC) of GO enrichment analysis and the target gene data of KEGG pathway were visualized by online drawing software of microbioinformatics (http: / / www.bioinformatics.com.cn / ). The related biological processes and key pathways of CEZY anti-AR were obtained. The top 10 pathways were visualized by Cytoscape3.7.2 software, and the KEGG core pathway was visualized by KEGG PATHWAY database (https: / / www.kegg.jp / kegg / pathway.html).
[0071] 2.6. Prediction results of potential target points of CEZY anti-AR: A total of 180 active ingredients of 8 traditional Chinese medicines in "Xiang'erzi drink" were obtained through TCMSP database and literature collection, and a total of 104 target points were predicted by Swiss Target Prediction. A total of 1397 target genes for regulating AR disease were obtained through Genecards, OMIM, and TTD databases. Figure 9As shown, mapping drug targets to disease targets yielded 119 overlapping targets. Figure 10 As shown.
[0072] 2.7. Results of "Disease-Drug-Ingredient-Intersecting Target" Network Analysis: Drugs, active ingredients, diseases, and intersecting targets were imported into Cytoscape 3.7.2 to construct a "Disease-Drug-Ingredient-Intersecting Target" network, as shown below. Figure 11 As shown. (Note: Arrows represent diseases, square nodes represent 8 Chinese herbal medicines, rhombus nodes represent the intersection target points of the 8 Chinese herbal medicines in CEZY and AR diseases, octagonal nodes represent the common active ingredients of the 8 Chinese herbal medicines, and circular nodes represent the effective active ingredients of each Chinese herbal medicine. Node size is distinguished according to the dgree value).
[0073] The network comprises 308 nodes and 1916 edges. Connections between nodes represent relationships between diseases and their corresponding targets, drugs and ingredients, and ingredients and their corresponding targets. Based on degree values, the top 10 active ingredients were selected, as shown in the CEZY anti-AR 10-key compound numbering table. Figure 12 As shown. These active ingredients are associated with multiple targets and are the main effective active ingredients of CEZY in the treatment of AR, such as quercetin, kaempferol, β-sitosterol, baicalin, and gentianin.
[0074] 2.8. Protein-Protein Interaction Network Analysis Results: 119 intersection target points were imported into the String database, and analysis yielded a "drug-disease" protein-protein interaction network (PPI) consisting of 119 nodes, 326 edges, an average node degree of 5.48, and an average local clustering coefficient of 0.542. Figure 13 As shown, different colors represent the degree value of a node. The node degree represents the number of times that node directly interacts with other nodes in the PPI network. The higher the degree, the more biological functions it participates in, and the stronger its biological importance. It can be seen that CEZY can regulate AR diseases through multiple pathways and multiple targets.
[0075] The aforementioned PPI network diagram has too many nodes and is quite complex, making it difficult to intuitively observe the relationships between the central nodes. The PPI network was exported as a .tsv file and imported into Cytoscape 3.7.2 software. The topology parameters were calculated, and core target points were selected based on Closeness > 0.348, Betweenness > 32.385, and Degree > 5, resulting in a core target network diagram consisting of 28 nodes and 126 edges. (See below for details.) Figure 14The color of the nodes in the network represents the degree value, with darker colors representing larger degree values and lighter colors representing smaller degree values. The top 11 target proteins in terms of degree value are TNF, STAT3, IL6, JUN, TP53, IL1B, AKT1, HSP90AA1, MAPK1, IFNG, and CXCL8, which are the core targets of CEZY anti-AR and are closely related to AR.
[0076] 2.9. GO function enrichment analysis results: GO function enrichment analysis was performed on the 119 intersection targets using the DAVID database, resulting in 576 biological process entries, 54 cellular component entries, and 145 molecular function entries. The top 10 results of GO-BP, GO-CC, and GO-MF were plotted using the micro-signal online analysis tool. As shown in Figure 15 , the top-ranked biological functions of GO enrichment are mainly RNA polymerase II-mediated transcriptional positive regulation, positive regulation of gene expression, signal transduction, and positive regulation of DNA template transcription. The top-ranked cellular components are mainly plasma membrane, cytoplasm, nucleus, and cytosol. The top-ranked molecular functions are mainly protein binding, homologous protein binding, enzyme binding, and protein homodimerization activity.
[0077] The top 10 results of KEGG enrichment pathways were plotted using the micro-signal online analysis tool. As shown in Figure 16 , the main pathways involved are cancer pathway, blood lipids and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, human cytomegalovirus infection, PI3K-Akt signaling pathway, chemical carcinogenesis-receptor activation, hepatitis B, IL-17 signaling pathway, and Chagas disease. The "core target-signal pathway" network was constructed using Cytoscape3.7.2, and it can be seen that the main targets involved are RELA, MAPK1, AKT1, IL6, JUN, IKBKB, MAPK14, TNF, BCL2, PRKCA, and CXCL8. As shown in Figure 17 (Notes: circles represent signal pathways, and diamonds represent core targets. The color depth and node size are distinguished according to the degree value).
[0078] The above functional enrichment analysis and "core target-signal pathway" network indicate that multiple core targets are significantly enriched in the PI3K-Akt signaling pathway.
[0079] III. In vitro experiments Objective: To preliminarily screen the anti-inflammatory and anti-allergic activities of each formula gel and explore its preliminary mechanism of action.
[0080] Experimental object: RBL-2H3 cells (a kind of rat basophilic leukemia cells, commonly used to simulate mast cell in type I allergic reaction).
[0081] Experimental steps: 3.1. Cell culture and sensitization: RBL-2H3 cells were cultured under appropriate conditions. The cells were divided into several groups: Blank control group: normal culture, no treatment.
[0082] Model group: stimulated with C48 / 80 (a mast cell degranulation inducer).
[0083] Positive drug group (such as cetirizine group): C48 / 80 stimulation + cetirizine solution.
[0084] Dosing group: C48 / 80 stimulation + different concentrations of formula one, two, three, basic optimized formula, primary monomer combination formula (M1, M2...), optimized ratio combination formula (P1, P2) warm sensitive gel solution.
[0085] 3.2. Index detection: β-amino hexosaminidase release rate determination: a marker of mast cell degranulation. Collect cell supernatant, detect using specific substrate, calculate release rate. The lower the release rate, the better the inhibition of degranulation.
[0086] Inflammatory factor detection: collect cell supernatant, detect the levels of histamine (Histamine), tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4) using ELISA kit.
[0087] Mean data table: the effect of each group on RBL-2H3 cell degranulation and inflammatory factor release ($\bar{x}\pms,n=6$), as shown in Figure 23 .
[0088] Experimental conclusion: in vitro experiments show that the warm sensitive gels of each level of optimized formula can effectively inhibit mast cell degranulation and release of inflammatory factors, and the effect gradually increases with the optimization steps. Formula five (P2) gel group has the most significant effect, better than the positive drug and all other formula groups, proving its strong anti-allergic and anti-inflammatory activity.
[0089] Four, animal model verification Experimental purpose: in vivo evaluation of the therapeutic effect of each level of formula five gel on allergic rhinitis (AR) animals, and in-depth exploration of its mechanism of action (such as AGE-RAGE pathway).
[0090] Experimental animals: formula five BALB / c mice or SD rats, half male and half female.
[0091] Experimental steps: 4.1. AR model establishment: Formula five used ovalbumin (OVA) sensitization method. Animals except the blank control group, were injected intraperitoneally with OVA, followed by OVA solution nasal provocation, for several weeks, to establish the AR model.
[0092] 4.2. Grouping and administration: Formula five randomly grouped the successfully modeled animals (same as in vitro experiment grouping), 10 in each group. The blank group and the model group were given saline nasal drops; the positive drug group was given cetirizine by gavage or a certain commercial nasal spray; each administration group was given Formula Three, the optimized base formula, Formula Four (M3), Formula Five (P2) Formula Five temperature-sensitive gel nasal drops, respectively. Administration was performed 30 minutes before each OVA provocation.
[0093] 4.3. Behavioral evaluation: Formula five recorded the number of times the animals scratched their noses, sneezed, and scratched their ears within 30 minutes after each provocation.
[0094] 4.4. Sample collection: After the last provocation, blood was taken, and serum was separated; the animals were sacrificed, and nasal mucosa tissue was taken.
[0095] 4.5 Index detection: Serological detection: Formula five used ELISA to detect OVA-specific IgE and histamine levels in serum.
[0096] Histopathological examination: After HE staining of the nasal mucosa tissue, the infiltration of inflammatory cells (such as eosinophils) was observed under a light microscope, and a pathological score was given.
[0097] Molecular biology detection (mechanism research): qRT-PCR: Formula five detected the mRNA expression levels of RAGE, NF-κB p65, TNF-α, and IL-4 in the nasal mucosa tissue.
[0098] Western Blot: Formula five detected the protein expression levels of RAGE, NF-κB p65, and phosphorylated NF-κB p65 (p-NF-κB p65) in the nasal mucosa tissue.
[0099] Mean data table: The effects of each group on the behavioral symptoms and serological indicators of AR model rats (Formula five $\bar{x}$ Formula five ± Formula five s, Formula five n = 10$), as shown in Figure 24 .
[0100] Results showed that compared with the model group, the protein expression levels of RAGE and p-NF-κB p65 in each administration group were down-regulated to different degrees. Among them, the down-regulation effect of the gel group of formula five (P2) was the most significant, almost close to the level of the blank control group, and the effect was better than that of all other formula groups and the positive drug group.
[0101] Experimental conclusion: behavior and serology: formula five (P2) temperature-sensitive gel can significantly alleviate the symptoms of AR animal rhinitis (sneezing, scratching), and reduce the level of allergic markers (IgE, histamine), and the effect is better than that of all previous formulas and positive drugs.
[0102] Histopathology: HE staining showed that a large number of inflammatory cell infiltration and damaged epithelial structure were observed in the nasal mucosa tissue of the model group. Each administration group was improved, and the inflammatory cell infiltration of the gel group of formula five (P2) was the least, and the tissue structure was the most complete, close to the normal state.
[0103] Mechanism of action: The temperature-sensitive gel prepared by the present application (especially formula five) may inhibit the AGE-RAGE signaling pathway, thereby inhibiting the activation of the downstream key factor NF-κB, and ultimately reducing the expression and release of various inflammatory factors, thereby treating allergic rhinitis. This verifies the prediction results of network pharmacology.
[0104] In summary, through rigorous in vitro and in vivo experiments, layer-by-layer screening and verification, it is finally proved that the temperature-sensitive gel drug delivery system prepared by the final monomer formula five is a safe, effective and mechanism-specific ideal drug for treating allergic rhinitis.
[0105] The specific embodiments described herein are merely illustrative of the spirit of the present application. Those skilled in the art of the present application can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, but will not deviate from the spirit of the present application or exceed the scope defined by the appended claims.
Claims
1. A method for preparing a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, comprising the following preparation steps: S1. Preliminary formulation of Xanthium sibiricum powder for treating rhinitis: Based on the existing formula, Formula 1, optimization will be carried out. Formula 1 consists of the following components by weight: The formula consists of 9 parts Xanthium sibiricum, 8 parts Angelica dahurica, 10 parts Magnolia biondii, and 5 parts Mentha haplocalyx. Based on clinical experience with traditional Chinese medicine, it has been optimized into Formula 2, which includes the following components in the indicated weight proportions: 9 parts Xanthium sibiricum, 8 parts Angelica dahurica, 10 parts Magnolia biondii, 5 parts Mentha haplocalyx, 5 parts Saposhnikovia divaricata, and 5 parts Astragalus membranaceus. S2. Raw material-level network pharmacology prediction: Network pharmacology was used to predict the core targets and pathways of Formula 2 in the treatment of allergic rhinitis. The extraction process and ratio of Formula 2 were further optimized by combining response surface methodology to obtain Formula 3. Formula 3 consists of the following components by weight: Xanthium sibiricum 9, Angelica dahurica 8, Magnolia biondii 10, Mentha haplocalyx 5, Saposhnikovia divaricata 5, Scutellaria baicalensis 8, Astragalus membranaceus 5, and Glycyrrhiza uralensis 6. S3. Trial production of raw material-grade thermosensitive gel products: Based on formula 1, formula 2 and formula 3, thermosensitive gel products were trial-produced respectively. A1. Prepare thermosensitive gel matrix; A2. Add the mixture of formula 1, formula 2 and formula 3 respectively to obtain several corresponding raw material-grade thermosensitive gel solutions; Formula 1 and formula 2 are used as control examples to prove that the efficacy of formula 3 is indeed better than either formula 1 or 2, thereby strengthening the demonstration of the inventiveness of the invention. S4. Raw material-level experimental testing: Multi-level experimental evaluation of the appearance, physical stability and rheological properties of several raw material-level thermosensitive gel solutions; in vitro experiments and animal model verification of the effectiveness in treating rhinitis; confirmation of the most effective raw material-level traditional Chinese medicine formula for treating rhinitis, which is recorded as the basic optimized formula; S5. Monomer-level network pharmacology prediction: The raw materials of traditional Chinese medicine in the basic optimized formula are replaced with active ingredient monomers that are determined by network pharmacology and response surface methodology, and several new formulas are formed by different types of active ingredient monomers. These are denoted as primary monomer combination formulas M1, M2, M3... S6. Preliminary trial production of monomer-grade thermosensitive gel products: Based on several primary monomer combination formulations M, thermosensitive gel products were trial produced. A1. Prepare thermosensitive gel matrix; B2. Add several primary monomer combination formulations M to the mixture to obtain several corresponding monomer-grade preliminary thermosensitive gel solutions. S7. Preliminary experimental testing of monomer-level solutions: multi-level experimental evaluation of the appearance, physical stability and rheological properties of several preliminary thermosensitive gel solutions of monomer-level solutions; in vitro experiments and animal model verification of the effectiveness in treating rhinitis; confirmation of the most effective monomer-level traditional Chinese medicine formula for treating rhinitis, which is referred to as Formula Four. S8. Formula Upgrade: The proportions of each effective ingredient in Traditional Chinese Medicine Formula 4 are adjusted, optimized, and upgraded to obtain several new formulas, denoted as optimized proportion combination formulas P1, P2, P3...; S9. Trial production of monomer-grade thermosensitive gel products: Trial production of thermosensitive gel products based on optimized ratio combination formula P: A1. Prepare thermosensitive gel matrix; C2. Add several mixtures of optimized ratio combination formula P respectively to obtain several corresponding monomer-grade thermosensitive gel solutions. S10. Monomer-level re-experimental testing: Multi-level experimental evaluation of the appearance, physical stability and rheological properties of several monomer-level re-thermosensitive gel solutions; in vitro experiments and animal model verification of the effectiveness in treating rhinitis; confirmation of the most effective traditional Chinese medicine formula for treating rhinitis, which is designated as Formula 5. Formula 5 is the final monomer formula, and the thermosensitive gel prepared based on it has the best properties and therapeutic effect.
2. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 1, is characterized in that... The formulation and preparation method of the thermosensitive gel matrix prepared by A1 are as follows: poloxamer 188, 1%, w / v; poloxamer 407, 20%, w / v; polyethylene glycol 6000, 1%, w / v; HP-β-CD, 3%, w / v; methylcellulose, 2%, w / v; and chitosan quaternary ammonium salt, 1%, w / v. These are dissolved and dispersed evenly in an appropriate amount of purified water to form the thermosensitive gel matrix.
3. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 2, is characterized in that... The method of adding A2, B2 and C2 to the mixture of each formulation is as follows: the mixture of each formulation and purified water are added to the thermosensitive gel matrix while stirring to obtain a mixed solution. The composition ratio of the mixture of each formulation is 72%, w / v.
4. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 3, is characterized in that... The method for preparing the thermosensitive gel solution from the mixed solution is as follows: the mixed solution is refrigerated at 4°C for more than 24 hours to swell, then restored to room temperature, and purified water is added to the prescribed amount to obtain the thermosensitive gel solution. The prescribed amount of purified water is the solution volume in terms of mass-volume concentration. The pH of the thermosensitive gel solution obtained from formula five is 5.6±0.
1.
5. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 4, is characterized in that... The thermosensitive gel solution prepared by Formula 5 has the following appearance: at 25°C, it is a homogeneous, free-flowing sol; when the temperature is raised to 32.6°C, a significant thermo-induced reversible phase transition occurs, forming a gel with elasticity and strong adhesion, indicating that its appearance meets the requirements.
6. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 5, is characterized in that... The physical stability of the thermosensitive gel solution prepared by Formula 5 was tested by centrifugation at 3000 r / min for 30 min. No layering, precipitation or phase separation was observed, indicating that it has good physical stability.
7. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 6, is characterized in that... The rheological properties of the thermosensitive gel in Formula 5 were tested using a rheometer. An appropriate amount of Xanthium sibiricum compound thermosensitive gel was placed on a PP25 stainless steel parallel plate with a diameter of 25 mm. The test gap parameter was set to 0.6 mm. The tests included gel linear viscoelastic region test, temperature scan test, frequency scan test and 3ITT test.
8. The preparation method of a traditional Chinese medicine delivery system based on Xanthium sibiricum formula for allergic rhinitis, as described in claim 7, is characterized in that... The T-test procedure consists of three stages: the first stage, 0.1% strain for 120s, low shear to simulate static state; the second stage, 100% strain for 40s, high shear to simulate drug administration shear; and the third stage, 0.1% strain for 300s, low shear to observe structural recovery; viscosity data are collected at 10 points / second in each stage.
9. A thermosensitive gel drug delivery system for treating allergic rhinitis, prepared by the method according to any one of claims 1-8.
10. The application of the thermosensitive gel drug delivery system as described in claim 9 in the preparation of a drug for treating allergic rhinitis, wherein a thermosensitive gel in a free-flowing sol state at room temperature of 25°C is placed inside the nasal cavity, and the breathing airflow raises the temperature of the thermosensitive gel to 32.6°C, causing a significant thermo-induced reversible phase transition, forming a gel state with a certain elasticity and strong adhesion. This transition can effectively resist ciliary clearance, significantly prolong the residence time of the drug at the site of action, and achieve sustained release.