Chewable tablet for enhancing immunity and preparation method thereof

Abstract

The invention provides a chewable tablet for enhancing immunity and a preparation method thereof. The chewable tablet can enhance immunity and prevent pollen allergy. The core of the method is that a chitosan-coated IL-4-siRNA and IL-13-siRNA compound is scientifically compatible with a sophora flower bud extract, and intervention is carried out from an early link of immunoregulation. The siRNA compound is used for specifically silencing gene expression of key cell factors IL-4 and IL-13 for driving anaphylaxis through an RNA interference technology, and an anaphylaxis signal is blocked from the source; the sophora flower bud extract is rich in flavonoid compounds, assists in inhibiting inflammatory reaction, and has synergistic interaction with siRNA to block anaphylactic reaction and enhance immunity.

Description

Technical Field

[0001] This application belongs to the field of health food, specifically relating to a chewable tablet that enhances immunity and its preparation method. Background Technology

[0002] With the fast pace of modern life and changes in the natural environment, the incidence of pollen allergy (seasonal allergic rhinitis) has increased significantly, becoming a common health problem affecting the quality of life of a wide range of people. Pollen allergy is essentially an abnormal response of the immune system to pollen allergens, with interleukins IL-4 and IL-13 being key cytokines driving this allergic reaction. Current common interventions, such as antihistamines or topical corticosteroids, mainly focus on relieving symptoms after they occur, while methods for preemptively regulating immunity and preventing allergies remain relatively limited.

[0003] In the health food industry, some products have attempted to achieve anti-allergy or immune-modulating functions through natural plant ingredients (such as sophora japonica extract and buckwheat sprouts). However, most of these products have broad mechanisms of action and lack precise intervention on the core pathways of allergies, thus their preventive efficacy is insufficient.

[0004] In recent years, advancements in biotechnology have brought new possibilities to the field of nutrition and health care. RNA interference technology can provide a new pathway for precise immune regulation by specifically silencing target genes. For example, using small interfering RNA to silence the expression of IL-4 and IL-13 can help the body maintain immune balance before allergy season, thereby preventing pollen allergy reactions. How to safely, stably, and conveniently apply this cutting-edge technology to daily health foods is becoming a key focus of the industry.

[0005] Therefore, the market urgently needs a daily preventative health food that integrates modern biotechnology with traditional natural ingredients, focusing on immune regulation and reducing pollen allergies. Summary of the Invention

[0006] To prevent pollen allergies and enhance immunity, this application aims to provide a chewable tablet that enhances immunity by scientifically combining an siRNA complex targeting IL-4 and IL-13 with functional ingredients such as Sophora japonica extract. This intervention targets the early stages of immune regulation and provides a safe chewable tablet for preventing pollen allergies and improving resistance through the precise delivery of the siRNA complex.

[0007] The purpose of this application is also to provide a method for preparing an immune-enhancing chewable tablet, which has mild conditions, simple process, and can effectively prepare an immune-enhancing chewable tablet.

[0008] To achieve the above objectives, this application provides an immune-enhancing chewable tablet and its preparation method, which is implemented using the following technical solution: On the one hand, this application provides a chewable tablet to enhance immunity, the chewable tablet being composed of the following raw materials: 10-12 parts by weight of starch, 20-40 parts by weight of type I collagen, 10-16 parts by weight of siRNA complex, 10-16 parts by weight of Sophora japonica extract, and 30-40 parts by weight of other excipients.

[0009] Preferably, the siRNA complex is chitosan-encapsulated siRNA, wherein the siRNA is IL-4-siRNA and IL-13-siRNA.

[0010] Preferably, the mass ratio of IL-4-siRNA to IL-13-siRNA is 1:1.

[0011] Preferably, the other excipients are 30-38 parts by weight of polysaccharide matrix and 0.5-2 parts by weight of sweetener.

[0012] Preferably, the polysaccharide matrix is ​​sodium alginate, pectin, and microcrystalline cellulose, and the sweetener is either xylitol or citrate.

[0013] This application provides an immune-enhancing chewable tablet, which comprises an siRNA complex consisting of IL-4-siRNA and IL-13-siRNA encapsulated by chitosan. Chitosan acts as a carrier to protect the siRNA from degradation and promote its absorption in the intestine, achieving precise targeted regulation. IL-4 and IL-13 are key cytokines driving pollen allergies. The siRNA specifically silences the gene expression of these two cytokines through RNA interference technology, blocking the initiation of the allergic reaction at its source. The Sophora japonica extract assists in immune regulation, and its flavonoid active ingredients can help inhibit the inflammatory response, working synergistically with the siRNA complex to enhance the immune-regulating effect.

[0014] On the other hand, this application provides a method for preparing an immune-enhancing chewable tablet, comprising the following steps: (1) Mix type I collagen powder at 20-40% w / w into 0.1% acetic acid and stir magnetically at 4-8℃ for 1-3 hours to obtain a uniform collagen solution. (2) Mix starch with deionized water (60℃) at 10-12% w / w and stir for 10 minutes to allow it to fully expand and form starch paste base; (3) Add sodium alginate, pectin and microcrystalline cellulose to deionized water in a ratio of 7:3:30 (w / w) and stir for 15 min to obtain a polysaccharide matrix suspension. (4) Add the starch paste base from step (2) to the polysaccharide matrix suspension from step (3) and continue stirring for 10 min to obtain a starch-polysaccharide mixed matrix; (5) Mix the collagen solution from step (1) and the starch-polysaccharide mixed matrix from step (4), add the siRNA complex and Sophora japonica extract, and homogenize in a stirring tank at 300 rpm to form a uniform wet mixture. Add sweetener and continue homogenizing to obtain a wet mixture. (6) The wet material from step (5) is spray-dried and then compressed with a rotary tablet press at a force of 20 kN to obtain chewable tablets that enhance immunity.

[0015] Preferably, in step (6), the spray drying is carried out at a feed temperature of 40℃-45℃ and a spray gas pressure of 0.8MPa for a drying time of 30min-45min.

[0016] Preferably, in step (5), the siRNA complex is prepared by dissolving chitosan in a 0.1% acetic acid system, adjusting the pH to 5.5, and preparing a 2% w / v chitosan solution; preparing a 0.5% w / v aqueous solution of sodium tripolyphosphate, denoted as TPP solution; adding the TPP solution dropwise to the chitosan solution under magnetic stirring at 800 rpm to form an ionic gel, and adding IL-4-siRNA and IL-13-siRNA to this system, and continuing to stir for 30 min to obtain the siRNA complex.

[0017] Preferably, in step (5), the preparation method of the Sophora japonica extract is as follows: take dried Sophora japonica flowers, crush them, sieve them, and extract them twice with 8-10 times the amount of 70% ethanol at 80°C for 1.5 hours each time; combine the extracts, filter and purify to obtain Sophora japonica extract.

[0018] More preferably, the sieving is performed through an 80-mesh sieve.

[0019] This application provides a method for preparing an immune-enhancing chewable tablet. Firstly, it employs targeted treatment of heat-sensitive components. Type I collagen is dissolved in 0.1% acetic acid at a low temperature of 4-8℃ to avoid damaging its amino acid chain structure at high temperatures, thus preserving its function in assisting immune regulation. Although the Sophora japonica extract is extracted by reflux extraction with ethanol at 80℃, the polarity of 70% ethanol and two extraction purification processes efficiently retain the anti-inflammatory active components of flavonoids while removing impurities. Uniform dispersion is achieved through stepwise matrix construction. First, starch swells in deionized water at 60℃ to form a paste base, which is then mixed with a polysaccharide matrix suspension prepared in a 7:3:30 ratio to form a stable starch-polysaccharide mixed matrix. Subsequently, collagen solution, siRNA complex, and... Sophora japonica extract was homogenized at 300 rpm to avoid activity conflicts or uneven dispersion caused by excessively high local component concentrations, laying the foundation for the consistency of efficacy in the final tablets. On the other hand, targeted delivery was achieved through the preparation of siRNA complexes using TPP ionogel technology: chitosan was dissolved in 0.1% acetic acid and adjusted to pH 5.5, and formed a gel with TPP solution under stirring at 800 rpm. Its cationic properties can tightly bind to IL-4-siRNA and IL-13-siRNA to form a protective shell, which not only prevents siRNA from being degraded by nucleases in the intestine, but also promotes the absorption of siRNA by epithelial cells by relying on the intestinal mucosal adhesion of chitosan, thereby achieving targeted silencing of key allergy cytokines.

[0020] The beneficial effects of the embodiments in this application are as follows: This application provides an immune-enhancing chewable tablet and its preparation method. The siRNA complex consists of IL-4-siRNA and IL-13-siRNA encapsulated by chitosan. Chitosan acts as a carrier to protect the siRNA from degradation and promote its absorption in the intestine, achieving precise targeted regulation. IL-4 and IL-13 are key cytokines driving pollen allergy. The siRNA specifically silences the gene expression of these two cytokines through RNA interference technology, blocking the initiation of the allergic reaction at its source. Experimental data show that the serum total IgE level in the model control group mice soared to 850.5. The serum IgE concentration was significantly higher than that of the normal control group (185.3 ng / mL), accompanied by severe eosinophilic infiltration in the lung tissue and airway hyperresponsiveness, successfully simulating the pathological state of allergic diseases. After drug intervention in each example group, especially in Example 2 group, the serum IgE concentration dropped to 215.6 ng / mL, which is very close to the normal level; the inflammatory infiltration in the lung tissue was significantly reduced, the eosinophil count dropped to 18.2 cells / HPF; and the airway hyperresponsiveness also basically returned to normal. This indicates that the chewable tablet can effectively block the core pathway of allergic reactions.

[0021] Meanwhile, Western blotting revealed its mechanism of action at the protein expression level. The results showed that without the protection of the chitosan delivery system, siRNA was completely inactivated via oral administration, demonstrating the protective effect of chitosan on siRNA. Sophora japonica extract, as an important auxiliary component, is rich in flavonoids and has strong anti-inflammatory and antioxidant activities. It can help inhibit the release of inflammatory mediators, reduce oxidative stress in the respiratory mucosa, and form a synergistic relationship with the siRNA complex to jointly block the complete chain of allergic reactions. Type I collagen can form microstructures in the intestine after oral administration, promote the integrity of the mucosal barrier, help maintain intestinal immune homeostasis, and provide a better physiological environment for the delivery and absorption of siRNA, further ensuring the efficacy of the core components.

[0022] Therefore, this chewable tablet enhances the immunomodulatory effect and prevents pollen allergies by combining a gene-silencing siRNA complex with the auxiliary immune regulation of Sophora japonica extract. The flavonoid active ingredients in Sophora japonica extract can help suppress the inflammatory response and work synergistically with the siRNA complex. Attached Figure Description

[0023] Figure 1 This is a statistical analysis chart of the serum total IgE concentration results in Experiment Example 2 of this application; Figure 2 This is a statistical analysis chart of the eosinophil count results in lung tissue from Experiment Example 2 of this application; Figure 3 This is a statistical analysis graph of the airway hyperresponsiveness results in Test Example 2 of this application; Figure 4 This is a Western blotting diagram from Experiment Example 2 of this application. Detailed Implementation

[0024] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0025] In the following examples, IL-4 and IL-13 were purchased from ThermoFisher; the BCA protein concentration assay kit was purchased from Beyotime Biotechnology; the IL-4 antibody was purchased from Abcam; the IL-13 antibody was purchased from Abcam; the β-actin antibody was purchased from CST; the secondary antibody (goat anti-rabbit) was purchased from Abbkine; and other raw materials and reagents not specifically mentioned were all available commercially.

[0026] Example 1 Chewable tablets to boost immunity (1) Ingredients: 10g starch, 20g type I collagen, 10g siRNA complex, 10g Sophora japonica extract, 30g polysaccharide matrix (5.25g sodium alginate, 2.25g pectin, 22.5g microcrystalline cellulose), 0.5g xylitol.

[0027] (2) Preparation method: 1) Preparation of siRNA complex: a. Dissolve 2% w / v chitosan in 0.1% acetic acid and adjust the pH to 5.5; b. Prepare a 0.5% w / v sodium tripolyphosphate (TPP) solution; c. Under magnetic stirring at 800 rpm, the TPP solution is added dropwise to the chitosan solution to form an ionic gel.

[0028] d. Add IL-4-siRNA and IL-13-siRNA (mass ratio 1:1) to the system and continue stirring for 30 min to obtain the siRNA complex; e. Characterization of the siRNA complex: its particle size is 80-150 nm and its encapsulation efficiency is >85%.

[0029] 2) Preparation of Sophora japonica flower extract: a. Drying: Place the purchased Sophora japonica flowers (dried Chinese medicinal material) in an 80℃ oven for 5 hours to prevent residual moisture from affecting the efficiency of subsequent pulverization and extraction, and to remove moisture and reduce pulverization resistance; b. Crushing: Use a plant crusher to coarsely crush the dried Sophora japonica flowers to a particle size of ≤2mm to increase the surface area and improve solvent penetration; c. Sieving: Pass the crushed coarse powder through an 80-mesh metal sieve, collect the fine powder that passes through, and recycle the residue for further crushing to ensure uniform particle size and avoid large pieces affecting the uniformity of reflux. d. Extraction: 70% ethanol was used as the extraction solvent, the reflux temperature was 80℃, and each extraction was performed for 90 min. After two extractions, the supernatants were combined to obtain the crude Sophora japonica extract. e. Purification: Transfer the combined supernatant to a 50 mL centrifuge tube, centrifuge at 8000 r / min for 10 min, collect the supernatant, remove fine suspended particles to prevent subsequent filtration clogging, and filter the supernatant through a 0.22 µm needle filter (PVDF) for a second time. Collect the filtrate in a clean glass bottle to obtain a clear extract, transfer it to a rotary evaporator, and concentrate it at a water bath temperature of 45℃-50℃. Place the concentrate in a refrigerator at 4℃ for 12 h to promote the precipitation of flavonoids. After refrigeration, centrifuge at 8000 r / min for 10 min, discard the supernatant, collect the precipitate, transfer it to a vacuum drying oven at 45℃, and dry for 7 h until constant weight is obtained to obtain Sophora japonica extract.

[0030] 3) Preparation of chewable tablets: a. Preparation of collagen solution: Type I collagen powder was added to 0.1% acetic acid solution at 20% w / w and magnetically stirred at 4℃ for 1 h to obtain a homogeneous collagen solution; b. Preparation of starch paste base: Mix starch at 10% w / w with deionized water at 60℃ and stir for 10 min to allow it to fully swell and form starch paste base; c. Preparation of polysaccharide matrix suspension: Sodium alginate, pectin and microcrystalline cellulose were added to deionized water according to the above weight, and stirred for 15 min to obtain polysaccharide matrix suspension; d. Preparation of starch-polysaccharide mixed matrix: Add starch paste base to polysaccharide matrix suspension, continue stirring for 10 min, and mix evenly; e. Preparation of wet material: The collagen solution was mixed with the starch-polysaccharide mixed matrix, the siRNA complex and Sophora japonica extract were added, and the mixture was homogenized at 300 rpm. Then xylitol was added and homogenization was continued to obtain the wet material. f. Tableting: The wet material is spray-dried (feed temperature 40℃, spray pressure 0.8MPa, drying time 30min), and then compressed into tablets using a rotary tablet press under a pressure of 20kN to obtain chewable tablets.

[0031] Example 2 Chewable tablets to boost immunity (1) Ingredients: 12g starch, 40g type I collagen, 16g siRNA complex, 16g Sophora japonica extract, 38g polysaccharide matrix (6.65g sodium alginate, 2.85g pectin, 28.5g microcrystalline cellulose), 2g citrate (Note that xylitol itself has a distinct sweetness, while citrate itself has no sweetness, but it can mask unpleasant flavors (EP2123166A1 has been disclosed), which is suitable for some elderly people, diabetic patients or people who are on a low-sugar diet, so citrate is used instead of xylitol in this embodiment).

[0032] (2) Preparation method: Same as in Example 1.

[0033] Example 3 Chewable tablets to boost immunity (1) Ingredients: 11g starch, 25g type I collagen, 12g siRNA complex, 12g Sophora japonica extract, 34g polysaccharide matrix (5.95g sodium alginate, 2.55g pectin, 25.5g microcrystalline cellulose), 1g xylitol.

[0034] (2) Preparation method: Same as in Example 1.

[0035] Example 4 Chewable tablets to boost immunity (1) Ingredients: 11g starch, 35g type I collagen, 14g siRNA complex, 15g Sophora japonica extract, 36g polysaccharide matrix (6.3g sodium alginate, 2.7g pectin, 27g microcrystalline cellulose), 1.5g citrate (Note that xylitol itself has a distinct sweetness, while citrate itself has no sweetness, but it can mask unpleasant flavors (EP2123166A1 has been disclosed), which is suitable for some elderly people, diabetic patients or people who are on a low-sugar diet, so citrate is used instead of xylitol in this embodiment).

[0036] (2) Preparation method: Same as in Example 1.

[0037] Comparative Example 1 Chewable tablets to boost immunity (siRNA-free) (1) Ingredients: 10g starch, 20g type I collagen, 10g sophora japonica extract, 30g polysaccharide matrix (5.25g sodium alginate, 2.25g pectin, 22.5g microcrystalline cellulose), 0.5g xylitol.

[0038] (2) Preparation method: 1) Preparation of Sophora japonica flower extract: Same as in Example 1; 2) Preparation of chewable tablets: a. Preparation of collagen solution: Type I collagen powder was added to 0.1% acetic acid solution at 20% w / w and magnetically stirred at 4℃ for 1 h to obtain a homogeneous collagen solution; b. Preparation of starch paste base: Mix starch at 10% w / w with deionized water at 60℃ and stir for 10 min to allow it to fully swell and form starch paste base; c. Preparation of polysaccharide matrix suspension: Sodium alginate, pectin and microcrystalline cellulose were added to deionized water according to the above weight, and stirred for 15 min to obtain polysaccharide matrix suspension; d. Preparation of starch-polysaccharide mixed matrix: Add starch paste base to polysaccharide matrix suspension, continue stirring for 10 min, and mix evenly; e. Preparation of wet material: The collagen solution was mixed with the starch-polysaccharide mixed matrix, the Sophora japonica extract was added, and the mixture was homogenized at 300 rpm. Then xylitol was added and homogenized again to obtain the wet material. f. Tableting: The wet material is spray-dried (feed temperature 40℃, spray pressure 0.8MPa, drying time 30min), and then compressed into tablets using a rotary tablet press under a pressure of 20kN to obtain chewable tablets.

[0039] Comparative Example 2 Chewable tablets to boost immunity (without Sophora japonica extract) (1) Ingredients: 10g starch, 20g type I collagen, 10g siRNA complex, 30g polysaccharide matrix (5.25g sodium alginate, 2.25g pectin, 22.5g microcrystalline cellulose), 0.5g xylitol.

[0040] (2) Preparation method: 1) Preparation of siRNA complex: Same as in Example 1; 2) Preparation of chewable tablets: a. Preparation of collagen solution: Type I collagen powder was added to 0.1% acetic acid solution at 20% w / w and magnetically stirred at 4℃ for 1 h to obtain a homogeneous collagen solution; b. Preparation of starch paste base: Mix starch at 10% w / w with deionized water at 60℃ and stir for 10 min to allow it to fully swell and form starch paste base; c. Preparation of polysaccharide matrix suspension: Sodium alginate, pectin and microcrystalline cellulose were added to deionized water according to the above weight, and stirred for 15 min to obtain polysaccharide matrix suspension; d. Preparation of starch-polysaccharide mixed matrix: Add starch paste base to polysaccharide matrix suspension, continue stirring for 10 min, and mix evenly; e. Preparation of wet material: The collagen solution was mixed with the starch-polysaccharide mixed matrix, the siRNA complex was added, and the mixture was homogenized at 300 rpm. Then xylitol was added and homogenization was continued to obtain the wet material. f. Tableting: The wet material is spray-dried (feed temperature 40℃, spray pressure 0.8MPa, drying time 30min), and then compressed into tablets using a rotary tablet press under a pressure of 20kN to obtain chewable tablets.

[0041] Comparative Example 3 Chewable tablets to boost immunity (siRNA uncoated) (1) Ingredients: 10g starch, 20g type I collagen, 10g siRNA, 10g Sophora japonica extract, 30g polysaccharide matrix (5.25g sodium alginate, 2.25g pectin, 22.5g microcrystalline cellulose), 0.5g xylitol.

[0042] (2) Preparation method: 1) Preparation of Sophora japonica flower extract: Same as in Example 1; 2) Preparation of chewable tablets: a. Preparation of collagen solution: Type I collagen powder was added to 0.1% acetic acid solution at 20% w / w and magnetically stirred at 4℃ for 1 h to obtain a homogeneous collagen solution; b. Preparation of starch paste base: Mix starch at 10% w / w with deionized water at 60℃ and stir for 10 min to allow it to fully swell and form starch paste base; c. Preparation of polysaccharide matrix suspension: Sodium alginate, pectin and microcrystalline cellulose were added to deionized water according to the above weight, and stirred for 15 min to obtain polysaccharide matrix suspension; d. Preparation of starch-polysaccharide mixed matrix: Add starch paste base to polysaccharide matrix suspension, continue stirring for 10 min, and mix evenly; e. Preparation of wet material: The collagen solution was mixed with the starch-polysaccharide mixed matrix, siRNA and Sophora japonica extract were added, and the mixture was homogenized at 300 rpm. Then xylitol was added and homogenization was continued to obtain the wet material. f. Tableting: The wet material is spray-dried (feed temperature 40℃, spray pressure 0.8MPa, drying time 30min), and then compressed into tablets using a rotary tablet press under a pressure of 20kN to obtain chewable tablets.

[0043] Comparative Example 4 Chewable tablets to boost immunity (1) Ingredients: 15g starch, 41g type I collagen, 18g siRNA complex, 18g Sophora japonica extract, 40g polysaccharide matrix (7g sodium alginate, 3g pectin, 30g microcrystalline cellulose), 2.5g xylitol.

[0044] (2) Preparation method: Same as in Example 1.

[0045] Comparative Example 5 Chewable tablets to boost immunity (1) Ingredients: 8g starch, 19g type I collagen, 9g siRNA complex, 8g Sophora japonica extract, 28g polysaccharide matrix (4.9g sodium alginate, 2.1g pectin, 21g microcrystalline cellulose), 0.5g xylitol.

[0046] (2) Preparation method: Same as in Example 1.

[0047] In accordance with the provisions of the Ministry of Health's "Technical Guidelines for Functional Testing and Evaluation of Health Foods (2025 Edition)," the chewable tablets prepared in the examples and comparative examples of this application were subjected to functional tests and the test results were evaluated.

[0048] Experimental Example 1 Chewable tablet appearance evaluation (1) After granulation of the above-mentioned examples and comparative examples, the appearance was evaluated, and the results are shown in Table 1: Table 1 Appearance Evaluation

[0049] (2) Based on the granulation appearance evaluation, Comparative Example 4 and Comparative Example 5 showed spots and were difficult to granulate, with the worst results.

[0050] Experimental Example 2 Pharmacological trials (1) Animal model: BALB / c female mice, 8 weeks old, weighing 18-22g, were provided by Jiangsu Hanjiang Biotechnology Co., Ltd.; OVA allergy model (subcutaneous injection of 20µg OVA + 2mg aluminum salt adjuvant on days 1 and 7; followed by nebulization of 1% OVA for 30min daily on days 14-18). This model can induce serum IgE, airway hyperresponsiveness (AHR) and eosinophil infiltration in lung tissue. All operations were carried out in accordance with the "Regulations on the Management of Experimental Animals". Anesthesia and euthanasia were performed using compliant drugs.

[0051] (2) Experimental groups: a total of 9 groups, 10 mice in each group: Example 1 group, Example 2 group, Example 3 group, Example 4 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Model group: given only an equal volume of physiological saline, Normal control group: mice that were not modeled; (3) Administration method: Oral gavage: once a day, the dosage for mice is 60 mg / kg / day. The chewable tablets are dissolved in physiological saline and administered by gavage until the end of the experiment (21 days in total). (4) Experimental methods: The serum total IgE concentration, lung eosinophil count, and airway hyperresponsiveness (Penh value) of mice in each group were measured. All experimental data were expressed as mean ± standard error (mean ± SEM), and were corrected by one-way ANOVA and Tukey's test. Compared with the model control, *p<0.05 was considered statistically significant, and **p<0.01 was considered statistically significant. The results are shown in Table 2, and the serum total IgE statistical graph is shown in Table 2. Figure 1 As shown, the eosinophil count in lung tissue is as follows. Figure 2 As shown in the figure, the airway hyperresponsiveness statistics are as follows: Figure 3 As shown: Table 2. Results of serum total IgE concentration, lung tissue eosinophil count, and airway hyperresponsiveness (Penh value) in mice.

[0052] Conclusion: The experimental results showed that the serum total IgE level in the model control group mice soared to 850.5 ng / mL, far exceeding the 185.3 ng / mL in the normal control group, accompanied by severe eosinophilic infiltration in the lung tissue and airway hyperresponsiveness, successfully simulating the pathological state of allergic disease. After drug intervention in each example group, especially in Example 2 group, the serum IgE concentration decreased to 215.6 ng / mL, which is very close to the normal level; the inflammatory infiltration in the lung tissue was significantly reduced, and the eosinophil count decreased to 18.2 cells / HPF; airway hyperresponsiveness also basically returned to normal. This indicates that the chewable tablet can effectively block allergic reactions. The core pathway; Comparative Group 3 (using uncoated naked siRNA) showed almost no difference in various indicators compared with the model control group, proving that without the protection of the chitosan delivery system, siRNA would be completely inactivated and unable to perform its gene silencing targeting function; Comparative Group 1 (without siRNA complex) showed only limited improvement, while Comparative Group 2 (without Sophora japonica extract) was better than Comparative Group 1, but still significantly weaker than the complete example group. The synergistic effect of siRNA complex and Sophora japonica extract: siRNA is responsible for precisely cutting off allergy signals at the source, while Sophora japonica extract, through its anti-inflammatory and antioxidant properties, helps to clear downstream inflammatory mediators.

[0053] It should be noted that a core characteristic of the Th2 immune response is that B cells produce a large number of IgE antibodies under the stimulation of cytokines such as IL-4 and IL-13, thereby triggering allergic symptoms. Experimental data showed that the serum total IgE level of mice in the model control group soared to 850.5 ng / mL, which was much higher than the 185.3 ng / mL of the normal control group. This directly reflects the presence of a strong Th2 immune response in the model group.

[0054] (6) Western Blot experiment 1) Sample collection and processing: Twenty-four hours after the last administration, mice were anesthetized and sacrificed. Lung tissue was rapidly separated, rinsed with pre-cooled physiological saline, flash-frozen in liquid nitrogen, and finally transferred to a -80°C freezer for storage. Protein extraction: Approximately 50 mg of lung tissue was taken, added to RIPA lysis buffer (containing protease inhibitor), homogenized on ice, centrifuged at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein concentration assay kit. Experiments were conducted on the normal control group, the model control group, Example 2, and Comparative Example 3. a. Electrophoresis: Take an equal amount of protein sample, mix it with the loading buffer, boil to denature it, and then perform SDS-PAGE gel electrophoresis to separate the protein; b. Transfer: Transfer the proteins separated by electrophoresis onto a PVDF membrane; c. Sealing: Seal with 5% skim milk powder at room temperature for 1 hour; d. Primary antibody incubation: Incubate overnight at 4°C with the following primary antibodies: anti-IL-4 antibody, anti-IL-13 antibody, and β-actin; e. Secondary antibody incubation: Incubate with the corresponding HRP-labeled secondary antibody at room temperature for 1 hour;

[0055] f. Development and quantification: Development is performed using ECL chemiluminescent reagent, and images are acquired through an imaging system.

[0056] 2) Results: The results are as follows Figure 4 As shown: β-actin: the bands are uniform in brightness and clear, and the positions are correct. The IL-4 and IL-13 bands in the normal control group are very weak, the bands in the model control group are bright, the bands in Example 2 are weak, and their brightness is close to that of the normal control group, indicating that protein expression has been successfully inhibited. The bands in Comparative Example 3 are very bright, close to that of the model control group.

[0057] (7) qPCR test: 1) Take 50mg of lung tissue from the normal control group, model control group, Example 2 and Comparative Example 3, extract RNA and perform qPCR to obtain the relative expression levels of IL-4 and IL-13. The Ct values ​​of the qPCR results are shown in Table 3 (each group was technically replicated three times and the average value was taken), the ΔCt values ​​are shown in Table 4 (ΔCt: target gene - internal reference), and the ΔΔCt values ​​are shown in Table 5 (ΔΔCt: ΔCt (experimental group) - ΔCt (normal control)). Table 3 Ct values ​​of qPCR results

[0058] Table 4 ΔCt values ​​of qPCR results

[0059] Table 5 ΔCt values ​​of qPCR results

[0060] 2) The results in Tables 3, 4 and 5 show that: Model control group: Compared with the normal control group, the relative expression levels of IL-4 and IL-13 mRNA in lung tissue will be significantly upregulated. Example 2 group: Compared with the model control group, the relative expression levels of IL-4 and IL-13 mRNA will be significantly downregulated, and it is expected to recover to levels close to those of the normal control group. Comparative example 3 group: The relative expression levels of IL-4 and IL-13 mRNA are not significantly different from those of the model control group and remain at a high level, proving that the chewable tablets can effectively silence the transcription of IL-4 and IL-13 genes in vivo through the siRNA complex.

[0061] Experimental Example 3 Long-term toxicity testing (1) Experimental animals: SPF-grade SD (Sprague-Dawley) rats, provided by Jiangsu Hanjiang Biotechnology Co., Ltd., half male and half female; age and weight: 6 weeks old, female weight 180-220g, male weight 220-260g; housing environment: temperature 20-26℃, relative humidity 40%-70%, 12h light and dark alternation, free access to food and water.

[0062] (2) Experimental grouping and administration: Rats were randomly divided into 4 groups, with 20 rats in each group (10 males and 10 females): Blank control group: given an equal volume of purified water; Low-dose group: Administer chewable tablets equivalent to 5 times the recommended human dose, dissolved in purified water; Medium dose group: Administer chewable tablets equivalent to 20 times the recommended human dose, dissolved in purified water; High-dose group: Administered chewable tablets equivalent to 50 times the recommended human dose, dissolved in purified water; Administration method: Oral gavage, once daily, for 6 consecutive months.

[0063] (3) Observation and detection indicators: 1) General clinical observation: Observe the appearance, behavior, activity, secretions and excrement of the animals daily, record mortality, weigh the animals weekly and record the amount of food consumed as shown in Table 6; Table 6 Clinical observation data

[0064] 2) Hematological examination: Blood was collected from the orbital venous plexus at 3 months, 6 months and the recovery period (4 weeks after drug withdrawal) to test indicators including red blood cell count, hemoglobin and platelet count. The results are shown in Table 7. Table 7 Blood Test Indicators

[0065] 3) Histopathological examination: After the experiment, all animals were euthanized and systematically autopsied. The organs were carefully observed for any abnormalities. The heart, liver, spleen, lungs, kidneys, adrenal glands, brain, testes / ovaries, stomach, intestines, and lymph nodes were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). The morphological changes were observed under an optical microscope, and the scores are shown in Table 8. Table 8 HE staining pathological scores

[0066] (4) Results: As shown in Tables 6, 7 and 8, when taken for a long period of time at doses far exceeding the recommended human dosage, the immune-enhancing chewable tablets of this application did not produce obvious toxic reactions in rats. All physiological and biochemical indicators remained within the normal range, and there was no pathological damage to the major organs, proving the safety of long-term consumption.

[0067] In summary, the core value of the immune-enhancing chewable tablets provided in this application lies in the innovative fusion of cutting-edge RNA interference technology with traditional natural extracts. Through a chitosan-encapsulated IL-4-siRNA and IL-13-siRNA complex, it achieves precise targeted intervention on IL-4 and IL-13 cytokines in the core allergy pathway, effectively inhibiting the overexpression of Th2-type immune responses at the gene level. Simultaneously, the addition of Sophora japonica extract with anti-inflammatory activity forms a synergistic mechanism of "source blocking" and "downstream assistance." Animal experimental data strongly confirm its efficacy: compared with the model control group, all example groups significantly reduced serum total IgE concentration, inhibited eosinophil infiltration in lung tissue, and improved airway hyperresponsiveness, with significantly better effects than the control group lacking the core component. Western blot results further intuitively demonstrate its powerful inhibitory ability on IL-4 and IL-13 protein expression. Therefore, this product not only provides a scientific, efficient, and safe daily health care solution for the prevention of pollen allergies, but also provides a framework and inspiration for the development of subsequent precision nutrition products.

[0068] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. An immunity-enhancing chewable tablet, characterized by comprising, The chewable tablet is composed of the following raw materials: starch 10-12 parts by weight, collagen type I 20-40 parts by weight, siRNA complex 10-16 parts by weight, sophora flower bud extract 10-16 parts by weight, and other auxiliary materials 30-40 parts by weight.

2. The chewable tablet for enhancing immunity as described in claim 1, characterized in that, The siRNA complex is chitosan-wrapped siRNA, wherein the siRNA is IL-4-siRNA and IL-13-siRNA.

3. The chewable tablet for enhancing immunity as described in claim 2, characterized in that, The mass ratio of the IL-4-siRNA to the IL-13-siRNA is 1:

1.

4. The chewable tablet for enhancing immunity as described in claim 1, characterized in that, The other auxiliary materials are 30-38 parts by weight of a polysaccharide matrix and 0.5-2 parts by weight of a sweetener.

5. The chewable tablet for enhancing immunity as described in claim 4, characterized in that, The polysaccharide matrix is sodium alginate, pectin, and microcrystalline cellulose, and the sweetener is any one of xylitol or citrate ester.

6. A method of preparing a chewable tablet for enhancing immunity, characterized in that, The preparation method is as follows: (1) collagen type I powder is mixed in 0.1% acetic acid at 20-40% w / w, and is stirred magnetically at 4-8°C for 1-3 hours to obtain a uniform collagen solution; (2) starch is mixed with 60°C deionized water at 10-12% w / w, and is stirred for 10 minutes to obtain a starch paste base; (3) sodium alginate, pectin, and microcrystalline cellulose are added to deionized water in a ratio of 7:3:30 (w / w), and are stirred for 15 minutes to obtain a polysaccharide matrix suspension; (4) the starch paste base in step (2) is added to the polysaccharide matrix suspension in step (3), and is stirred for another 10 minutes to obtain a starch-polysaccharide mixed matrix; (5) the collagen solution in step (1) and the starch-polysaccharide mixed matrix in step (4) are mixed, and the siRNA complex and sophora flower bud extract are homogenized in a stirring tank with a rotation speed of 300 rpm to form a uniform wet mixture, and a sweetener is added, and the wet mixture is obtained by further homogenization; (6) the wet mixture is subjected to spray drying, and then a rotary tablet press is used to obtain the chewable tablet for enhancing immunity at a tablet pressing force of 20 kN.

7. The method for preparing an immune-enhancing chewable tablet as described in claim 6, characterized in that, In step (6), the spray drying is performed at a feed temperature of 40-45°C and a spray gas pressure of 0.8 MPa, and the drying time is 30-45 minutes.

8. The method for preparing an immune-enhancing chewable tablet as described in claim 6, characterized in that, In step (5), the preparation method of the siRNA complex is as follows: chitosan is dissolved in a 0.1% acetic acid system, the pH is adjusted to 5.5, and a 2% w / v chitosan solution is prepared; sodium tripolyphosphate is prepared into a 0.5% w / v aqueous solution, which is recorded as a TPP solution; the TPP solution is added dropwise to the chitosan solution under magnetic stirring at a rotation speed of 800 rpm to form an ionic gel, and IL-4-siRNA and IL-13-siRNA are added to the system, and stirring is continued for 30 minutes to obtain the siRNA complex.

9. The method for preparing an immune-enhancing chewable tablet as described in claim 6, characterized in that, In step (5), the preparation method of the sophora flower bud extract is as follows: dried sophora flower buds are crushed, sieved, and extracted with 8-10 times the amount of 70% ethanol at 80°C for 2 times, each for 1.5 hours; the extraction solutions are combined, filtered, and purified to obtain the sophora flower bud extract.

10. The method for preparing an immune-enhancing chewable tablet as described in claim 9, characterized in that, The sieving is through an 80-mesh sieve.

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