A body immunity preparation for promoting HPV virus genome methylation

Abstract

This application relates to the field of biomedical technology, specifically disclosing an immune-boosting preparation that promotes HPV viral genome methylation. The preparation comprises epigallocatechin gallate, folic acid, vitamin C, vitamin E, zinc gluconate, various B vitamins, and excipients. Its preparation method involves pretreatment of the raw materials and excipients, microencapsulation of some vitamins, staged mixing via gradient shearing, dry granulation, low-temperature vacuum staged drying, and granulation to produce an oral solid dosage form. This preparation can be used to intervene in HPV infection, achieving dual intervention by promoting HPV viral genome methylation and enhancing the body's immunity. It has the advantages of a rational formulation and stable active ingredients; its preparation process effectively avoids oxidative inactivation of active ingredients, ensuring the uniformity of the preparation, and is simple to operate, suitable for large-scale production, and can meet the needs of clinical and routine HPV infection intervention.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and more specifically, to an immune agent that promotes the methylation of the HPV virus genome. Background Technology

[0002] HPV infection in women is a common clinical health problem. Persistent HPV infection can adversely affect women's health, creating a real need for safe and targeted HPV infection intervention agents. Most existing HPV-related conditioning agents primarily focus on simply boosting the body's immunity. Some products containing natural active ingredients are not designed with targeted formulations around HPV viral genome methylation, and the various components lack a reasonable compatibility logic, failing to form a synergistic system.

[0003] The core technical problem with current similar formulations lies in their inability to simultaneously achieve the dual synergistic effect of promoting HPV viral genome methylation and improving the body's immunity. A single mechanism of action cannot provide a comprehensive and effective intervention for HPV infection in women. The immune-boosting formulation that promotes HPV viral genome methylation described in this application aims to address the problem of existing formulations having a single mechanism of action and failing to achieve a synergistic effect of targeted methylation and immune regulation. It achieves a comprehensive intervention effect through a scientifically formulated compound. Summary of the Invention

[0004] To address the problem that existing formulations of the same type cannot simultaneously achieve the synergistic effect of promoting HPV viral genome methylation and enhancing the body's immunity, this application provides an immune-enhancing formulation that promotes HPV viral genome methylation.

[0005] In a first aspect, this application provides an immune-boosting agent that promotes HPV viral genome methylation, employing the following technical solution:

[0006] An immune-boosting agent that promotes HPV viral genome methylation, comprising the following raw materials in parts by weight: 140-160 parts epigallocatechin gallate; 0.3-0.8 parts folic acid; 20-250 parts vitamin C1; 25-50 parts vitamin E; 10-30 parts zinc gluconate; 5-12 parts vitamin B1; 2-5 parts vitamin B2; 2-5 parts vitamin B6; 0.005-0.01 parts vitamin B12; and 60-200 parts excipients.

[0007] By adopting the above technical solution, with epigallocatechin gallate as the core active substance, it can directly act on the HPV viral genome, initiate methylation-related pathways, and promote viral genome methylation. Folic acid can act as a methyl donor, providing necessary material support for the methylation process and assisting in enhancing the methylation-inducing effect of epigallocatechin gallate. Vitamin C and vitamin E work synergistically to exert antioxidant effects, reducing the oxidative loss of active ingredients. Zinc gluconate can regulate the metabolism and activity of immune cells in the body. Multiple B vitamins participate in the body's metabolic processes, providing a guarantee for immune function regulation. The raw materials are combined in specific weight proportions to form a synergistic system. Through the synergistic cooperation of multiple components, the dual effects of methylation induction and immune regulation are achieved. The excipients provide carriers for each active ingredient, ensuring the formation and stability of the formulation.

[0008] Preferably, the purity of the epigallocatechin gallate is 98.0%-99.5%.

[0009] By adopting the above technical solution and controlling the high purity range of epigallocatechin gallate, the interference of impurities in the crude product on its activity can be effectively reduced, and adverse interactions between impurities and other active ingredients can be avoided. This ensures that the core component can stably play its role in promoting HPV virus genome methylation. At the same time, high-purity epigallocatechin gallate is more likely to form a uniform mixing system with other raw materials, ensuring the consistency of the performance of each batch of the formulation and providing a foundation for the stable efficacy of the formulation.

[0010] Preferably, the excipients are selected from one or more of microcrystalline cellulose, lactose, croscarmellose sodium, magnesium stearate, and povidone K30.

[0011] By adopting the above technical solution, all selected excipients are pharmaceutical-grade, possessing good biocompatibility and will not chemically react with the active ingredients in the formulation, nor will they have adverse effects on the body. Among them, microcrystalline cellulose exhibits good flowability and compressibility, improving the formulation's molding effect; lactose improves the formulation's solubility, facilitating absorption by the body; croscarmellose sodium enhances the formulation's disintegration properties, promoting rapid release of the active ingredients; magnesium stearate acts as a lubricant, reducing friction during the formulation molding process; and povidone K30 acts as a binder, improving particle stability. Selecting appropriate excipient combinations based on different formulation forms can further optimize the formulation's molding quality and performance.

[0012] Preferably, the formulation is an oral solid dosage form, including tablets, capsules, or granules.

[0013] By adopting the above technical solutions, oral solid dosage forms require no special drug delivery equipment, are convenient to use, easy to carry and store, and suitable for long-term use. Tablets provide accurate dosage and are easy to take after molding; capsules can mask the odor of active ingredients, reduce gastrointestinal irritation, and protect the active ingredients from degradation in the stomach; granules allow for dosage adjustment as needed, making them suitable for different populations. These various formulations meet the needs of different usage scenarios and populations, improving the applicability of the formulations.

[0014] Secondly, this application provides a method for preparing an immune-boosting agent that promotes HPV viral genome methylation, employing the following technical solution:

[0015] A method for preparing an immune-boosting agent that promotes HPV viral genome methylation includes the following steps:

[0016] S1. Pretreatment of raw materials and auxiliary materials: Epigallocatechin gallate, as well as vitamin C, vitamin E, zinc gluconate, folic acid, vitamin B1, vitamin B2, vitamin B6 and vitamin B12 are sieved separately to obtain fine powder for later use.

[0017] S2, Vitamin Microencapsulation: A portion of the vitamin C obtained in step S1 is mixed with vitamin E, a portion of vitamin B1, vitamin B2, and vitamin B6, and then microencapsulated using ethyl cellulose aqueous dispersion as the coating material to obtain vitamin microcapsule particles.

[0018] S3. Gradient shear mixing: Epigallocatechin gallate, folic acid, zinc gluconate obtained in step S1, the remaining vitamin B1, vitamin B2, vitamin B6, and all vitamin B12 from step S2 are placed into a multi-dimensional motion mixer for first-gradient mixing; then the vitamin microcapsule granules prepared in step S2 and the remaining vitamin C are added for second-gradient mixing; finally, excipients are added for third-gradient mixing to obtain the final mixture.

[0019] S4. Formulation: The total mixture obtained in step S3 is granulated by dry granulation to obtain granules with uniform particle size.

[0020] S5. Drying and granulation: The granules obtained in step S4 are subjected to low-temperature vacuum drying to control the moisture content of the granules to below 2%, and then granulated to obtain the formulation.

[0021] By adopting the above technical solutions, the raw materials and excipients are pre-treated and sieved to remove impurities and ensure uniform particle size, laying the foundation for subsequent mixing and molding. Microencapsulation of some vitamins, utilizing the coating layer formed by ethyl cellulose aqueous dispersion, effectively isolates oxygen and moisture from the outside air, reducing oxidative degradation of vitamin components and extending the shelf life of the formulation. Gradient shear mixing, through staged mixing under different conditions, solves the problem of uneven mixing caused by differences in the solubility and density of different active ingredients, ensuring uniform distribution of each component in the total mixture. Dry granulation eliminates the need for added water, preventing water from damaging water-sensitive and heat-sensitive components such as epigallocatechin gallate and vitamins, thus ensuring the integrity of the active ingredients. Low-temperature vacuum drying quickly removes moisture from the granules at lower temperatures, while avoiding inactivation of active ingredients due to high temperatures. Granulation ensures uniform particle size, improving the molding quality and stability of the formulation.

[0022] Preferably, in step S1, the sieve mesh size is 60-100 mesh; for vitamin C, vitamin E and B vitamins, low-temperature pulverization is performed before sieving, and sieving is performed after pulverization. The pulverization temperature is 0-10℃ to obtain fine powder with uniform particle size.

[0023] By adopting the above technical solutions and controlling the sieve mesh size to 60-100 mesh, the particle size of the raw and auxiliary materials can be ensured to be moderate. This facilitates uniform subsequent mixing and guarantees the dissolution performance of the formulation after molding. It avoids uneven mixing or slow dissolution caused by excessively large particle size, while excessively small particle size easily leads to agglomeration. Low-temperature pulverization of vitamin components at 0-10℃ can effectively reduce the temperature rise during the pulverization process, preventing the vitamin components from undergoing oxidative degradation due to high temperatures and preserving their activity to the greatest extent. At the same time, low-temperature pulverization can make the vitamin powder particle size more uniform, further improving the effect of subsequent mixing and microencapsulation.

[0024] Preferably, in step S2, the parameters for microencapsulation are: inlet air temperature 40-55℃, material temperature 30-40℃, atomization pressure 0.15-0.25MPa, and coating weight gain controlled at 5%-15%.

[0025] By adopting the above technical solution and controlling the inlet air temperature and material temperature within a suitable range, it is possible to ensure that the ethyl cellulose aqueous dispersion can be uniformly atomized and form a complete coating layer on the surface of vitamin particles. This avoids excessively high temperatures that could lead to vitamin activity loss or coating layer charring, while excessively low temperatures would affect the drying speed and integrity of the coating layer. Appropriate atomization pressure allows the coating material to be atomized into uniform micro-droplets, ensuring a uniform coating layer thickness. With the coating weight gain controlled at 5%-15%, the coating layer can effectively isolate the external environment without affecting the vitamin release rate due to excessive thickness, thus achieving effective protection and controlled release of vitamins.

[0026] Preferably, in step S3, the mixing time of the first gradient mixing is 5-10 minutes and the mixing speed is 50-100 rpm; the mixing time of the second gradient mixing is 10-20 minutes and the mixing speed is 30-60 rpm; the mixing time of the third gradient mixing is 15-30 minutes and the mixing speed is 20-40 rpm; the ambient temperature is controlled at 15-25℃ and the relative humidity is controlled at 30-50% during the mixing process.

[0027] By employing the above technical solutions, the first gradient, using a higher rotation speed and shorter time, can quickly and evenly mix components with similar densities, such as epigallocatechin gallate, folic acid, and zinc gluconate. The second gradient, by reducing the rotation speed and extending the time, avoids damage to the vitamin microcapsule particles caused by high-speed stirring, while ensuring uniform mixing of the microcapsule particles with the remaining vitamin C. The third gradient, by further reducing the rotation speed and extending the time, ensures that the excipients are fully integrated with the previously mixed materials, avoiding uneven mixing caused by excipient agglomeration. Controlling the ambient temperature and relative humidity during the mixing process can prevent oxidation of active ingredients due to excessively high temperatures, and avoid excessive humidity causing the raw materials to absorb moisture and clump, affecting the mixing effect and formulation stability.

[0028] Preferably, in step S4, the process parameters for dry granulation are: feeding speed 20-40 rpm, roller speed 3-8 rpm, roller pressure 5-15 MPa, and granulation speed 100-200 rpm.

[0029] By adopting the above technical solutions, controlling the feeding speed, roller speed, roller pressure, and granulation speed within a suitable range ensures that the total mixed material can enter the space between the rollers evenly, forming tablets with uniform density. This avoids material accumulation and uneven pressure on the rollers due to excessive feeding, while excessively slow feeding will affect granulation efficiency. Appropriate roller pressure ensures that the granules have good hardness and wear resistance, preventing granules from being too brittle or too compact, which could affect dissolution. Matching the granulation speed with the roller speed and feeding speed ensures uniform particle size, avoiding excessively large or small particles and guaranteeing the consistency of the formulation's quality.

[0030] Preferably, in step S5, the drying temperature of the low-temperature vacuum drying is 30-45℃, the vacuum degree is controlled between -0.06MPa and -0.09MPa, and the drying time is 30-60 minutes; the drying adopts a segmented drying method, first drying at 30-35℃ for 20-30 minutes, and then drying at 40-45℃ for 20-30 minutes.

[0031] By adopting the above technical solution and controlling the drying temperature at 30-45℃, moisture in the granules can be effectively removed without deactivating the active ingredients. Vacuum control at -0.06MPa to -0.09MPa lowers the boiling point of water, accelerates the drying speed, shortens the drying time, and reduces the oxidative impact of air on the active ingredients. Segmented drying first removes free moisture from the granule surface at a lower temperature, then removes bound moisture from the granules at a slightly higher temperature. This balances drying efficiency with avoiding surface charring or active ingredient deactivation caused by single high-temperature drying. Furthermore, controlling the granule moisture content below 2% effectively prevents moisture absorption and clumping, improving the storage stability of the formulation.

[0032] In summary, this application has the following beneficial effects:

[0033] 1. Because this application uses a specific weight proportion of high-purity epigallocatechin gallate as the core active ingredient, and is combined with a scientifically formulated ratio of folic acid, vitamin C, vitamin E, zinc gluconate and various B vitamins, the active ingredients form a synergistic system that can effectively promote HPV virus genome methylation. At the same time, the vitamin components and zinc gluconate work together to enhance the body's immunity, thus achieving a comprehensive effect of treating female HPV virus infection through a dual mechanism of action.

[0034] 2. In this application, microcrystalline cellulose, lactose, croscarmellose sodium cellulose, magnesium stearate, and povidone K30 are preferred as excipients. These excipients have good flowability and compressibility, can be uniformly mixed with the active ingredients, and maintain stable physicochemical properties in the dry granulation process. They will not react adversely with the active ingredients, thus achieving the beneficial effects of good formulation formability and high stability of active ingredients.

[0035] 3. The preparation process of this application involves microencapsulating a portion of vitamin C, vitamin E, and B vitamins, using ethyl cellulose aqueous dispersion as the coating material to form vitamin microcapsule particles. This effectively isolates the vitamins from oxygen and moisture in the external air, slowing down the oxidative degradation of vitamin components during storage. Therefore, it achieves the technical effect of significantly improving the stability of the formulation and extending the shelf life of the product.

[0036] 4. The preparation process of this application adopts a gradient shear mixing method. Epigallocatechin gallate, folic acid, zinc gluconate and the remaining B vitamins are mixed in the first gradient. Then, vitamin microcapsule particles and the remaining vitamin C are added for the second gradient mixing. Finally, excipients are added for the third gradient mixing. By mixing in stages, the technical problem of uneven mixing of high dose vitamin C and trace amounts of B vitamins is solved. Therefore, the beneficial effect of uniform distribution and content uniformity of each component in the preparation is obtained.

[0037] 5. The method of this application adopts dry granulation combined with low-temperature vacuum segmented drying process. The whole process avoids contact with water and high temperature damage, which can maximize the preservation of the bioactivity of epigallocatechin gallate and heat-sensitive and water-sensitive components of vitamins. Therefore, it achieves the technical effect of effectively preserving the activity of active ingredients while ensuring good particle formation of the formulation. Attached Figure Description

[0038] Figure 1 This is a flowchart of a method for preparing an immune agent that promotes HPV viral genome methylation, as provided in this application. Detailed Implementation

[0039] The present application will be further described in detail below with reference to embodiments and comparative examples. Unless otherwise specified, the experimental methods used below are conventional methods. Unless otherwise specified, the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art, which can be obtained by those skilled in the art through commercial channels or prepared according to literature methods.

[0040] Technical Concept: Existing formulations targeting HPV infection in the biomedical field suffer from a single mechanism of action and insufficient synergy. The core reason lies in the lack of a scientifically sound compound formulation. Either they focus only on the effect of a single active ingredient, failing to simultaneously achieve the induction of viral genome methylation and the enhancement of the body's immunity in the biomedical field, or the component combinations lack logic, resulting in insufficient purity of the core active ingredient and the absence of effective active ingredient protection measures during preparation. This leads to the loss of easily oxidized components such as vitamins and uneven mixing of components, thereby affecting the overall efficacy and stability of the formulation and making it difficult to meet the actual needs of HPV infection intervention in the biomedical field.

[0041] This technical solution addresses the aforementioned issues through targeted design of compound formulations and preparation processes. The formulation uses high-purity epigallocatechin gallate as the core active ingredient, combined with folic acid, multiple vitamins, and zinc gluconate to form a synergistic system, providing material support for methylation induction and immune regulation. The process employs low-temperature pretreatment of raw materials and excipients, vitamin microencapsulation, gradient shear mixing, dry granulation, and low-temperature vacuum segmented drying to effectively protect the active ingredients from oxidation and inactivation, ensuring uniform mixing of all components and guaranteeing the quality and efficacy stability of the formulation. Ultimately, this achieves the dual effects of promoting viral genome methylation and enhancing the body's immunity in the field of HPV biomedical technology.

[0042] Preparation Example 1: The preparation method of high purity epigallocatechin gallate is as follows: Take 100 parts of crude tea polyphenol powder, of which the content of epigallocatechin gallate is 50%, add 700 parts of purified water, stir until completely dissolved, and prepare a tea polyphenol aqueous solution with a mass concentration of about 14%. Remove insoluble impurities by plate and frame filtration to obtain a clear loading solution.

[0043] The clarified sample solution was passed through a chromatography column packed with D101 macroporous adsorption resin at a flow rate of 1.8 column volumes per hour. After loading, the sample was first washed with 3000 parts of purified water at a flow rate of 2.5 column volumes per hour to remove water-soluble impurities such as sugars and proteins. Then, 2000 parts of 28% ethanol aqueous solution were used for elution at a flow rate of 2 column volumes per hour. The target eluent was collected in fractions and concentrated under reduced pressure until no alcohol odor was detected to obtain the crude extract of epigallocatechin gallate.

[0044] The crude extract was added to 800 parts of a 32% (v / v) methanol aqueous solution and stirred to dissolve, preparing a test solution with a mass concentration of about 12%. The solution was purified using a simulated moving bed chromatography system. The stationary phase was octadecylsilane-bonded silica gel, and the mobile phase was a 36% (v / v) methanol aqueous solution. The system switching time was set to 10 minutes, and the elution flow rate was controlled at 12 mL per minute. The target purified elution fraction was collected.

[0045] The target refined component was purified by vacuum distillation to remove the organic solvent. The concentrate was then transferred to a freeze dryer and freeze-dried at -38°C and a vacuum of -0.085 MPa for 9 hours to obtain a light yellow powder product.

[0046] The purity of epigallocatechin gallate in the product was 98.9% as determined by high performance liquid chromatography.

[0047] Example 1: This example provides an immune-boosting preparation that promotes HPV viral genome methylation, comprising the following raw materials in parts by weight: 150 parts epigallocatechin gallate, 0.55 parts folic acid, 85 parts vitamin C1, 37.5 parts vitamin E, 20 parts zinc gluconate, 8.5 parts vitamin B1, 3.5 parts vitamin B2, 3.5 parts vitamin B6, 0.0075 parts vitamin B12, and 130 parts excipients;

[0048] Epigallocatechin gallate was prepared using Preparation Example 1 with a purity of 98.75%; microcrystalline cellulose was used as the excipient; and the formulation was an oral solid dosage form, specifically a tablet.

[0049] The preparation method of the above-mentioned immune agent that promotes HPV viral genome methylation includes the following steps:

[0050] S1. Pretreatment of raw materials and auxiliary materials: Epigallocatechin gallate obtained in Preparation Example 1, as well as vitamin C, vitamin E, zinc gluconate, folic acid, vitamin B1, vitamin B2, vitamin B6 and vitamin B12, are sieved to obtain fine powder for later use.

[0051] The sieve mesh size is 80 mesh. For vitamin C, vitamin E and B vitamins, low-temperature pulverization is performed before sieving, and then sieving is performed after pulverization. The pulverization temperature is 5℃ to obtain fine powder with uniform particle size.

[0052] S2, Vitamin Microencapsulation: A portion of the vitamin C obtained in step S1 is mixed with vitamin E, a portion of vitamin B1, vitamin B2, and vitamin B6, and then microencapsulated using ethyl cellulose aqueous dispersion as the coating material to obtain vitamin microcapsule particles.

[0053] The parameters for microencapsulation are: inlet air temperature 47.5℃, material temperature 35℃, atomization pressure 0.2MPa, and coating weight gain controlled at 10%.

[0054] S3. Gradient shear mixing: Epigallocatechin gallate, folic acid, zinc gluconate obtained in step S1, the remaining vitamin B1, vitamin B2, vitamin B6, and all vitamin B12 from step S2 are placed into a multi-dimensional motion mixer for first-gradient mixing; then the vitamin microcapsule granules prepared in step S2 and the remaining vitamin C are added for second-gradient mixing; finally, excipients are added for third-gradient mixing to obtain the final mixture.

[0055] The mixing time for the first gradient mixing is 7.5 minutes and the mixing speed is 75 rpm; the mixing time for the second gradient mixing is 15 minutes and the mixing speed is 45 rpm; the mixing time for the third gradient mixing is 22.5 minutes and the mixing speed is 30 rpm; the ambient temperature is controlled at 20℃ and the relative humidity is controlled at 40% during the mixing process.

[0056] S4. Formulation: The total mixture obtained in step S3 is granulated by dry granulation to obtain granules with uniform particle size.

[0057] The process parameters for dry granulation are as follows: feeding speed 30 rpm, roller speed 5.5 rpm, roller pressure 10 MPa, and granulation speed 150 rpm.

[0058] S5. Drying and granulation: The granules obtained in step S4 are dried under low temperature vacuum to control the moisture content of the granules to below 2%, and then granulated to obtain the formulation.

[0059] The low-temperature vacuum drying process involves a drying temperature of 37.5℃, a vacuum level controlled at -0.075MPa, and a drying time of 45 minutes. The drying process is carried out in stages, first drying at 32.5℃ for 25 minutes, and then drying at 42.5℃ for 25 minutes.

[0060] Example 2: This example provides an immune-boosting preparation that promotes HPV viral genome methylation, comprising the following raw materials in parts by weight: 140 parts epigallocatechin gallate, 0.3 parts folic acid, 20 parts vitamin C1, 5 parts vitamin E2, 10 parts zinc gluconate, 5 parts vitamin B1, 2 parts vitamin B2, 2 parts vitamin B6, 0.005 parts vitamin B12, and 60 parts excipients;

[0061] Epigallocatechin gallate was prepared using Preparation Example 1 with a purity of 98.0%; lactose was used as the excipient; and the formulation was an oral solid dosage form, specifically a capsule.

[0062] The preparation method of the above-mentioned immune agent that promotes HPV viral genome methylation includes the following steps:

[0063] S1. Pretreatment of raw materials and auxiliary materials: Epigallocatechin gallate obtained in Preparation Example 1, as well as vitamin C, vitamin E, zinc gluconate, folic acid, vitamin B1, vitamin B2, vitamin B6 and vitamin B12, are sieved to obtain fine powder for later use.

[0064] The sieve used for sieving has a mesh size of 60. For vitamins C, E, and B, a low-temperature pulverization process is used before sieving, followed by sieving at a pulverization temperature of 0°C to obtain fine powder with uniform particle size.

[0065] S2, Vitamin Microencapsulation: A portion of the vitamin C obtained in step S1 is mixed with vitamin E, a portion of vitamin B1, vitamin B2, and vitamin B6, and then microencapsulated using ethyl cellulose aqueous dispersion as the coating material to obtain vitamin microcapsule particles.

[0066] The parameters for microencapsulation are: inlet air temperature 40℃, material temperature 30℃, atomization pressure 0.15MPa, and coating weight gain controlled at 5%.

[0067] S3. Gradient shear mixing: Epigallocatechin gallate, folic acid, zinc gluconate obtained in step S1, the remaining vitamin B1, vitamin B2, vitamin B6, and all vitamin B12 from step S2 are placed into a multi-dimensional motion mixer for first-gradient mixing; then the vitamin microcapsule granules prepared in step S2 and the remaining vitamin C are added for second-gradient mixing; finally, excipients are added for third-gradient mixing to obtain the final mixture.

[0068] The mixing time for the first gradient mixing is 5 minutes and the mixing speed is 50 rpm; the mixing time for the second gradient mixing is 10 minutes and the mixing speed is 30 rpm; the mixing time for the third gradient mixing is 15 minutes and the mixing speed is 20 rpm; the ambient temperature is controlled at 15℃ and the relative humidity is controlled at 30% during the mixing process.

[0069] S4. Formulation: The total mixture obtained in step S3 is granulated by dry granulation to obtain granules with uniform particle size.

[0070] The process parameters for dry granulation are as follows: feeding speed 20 rpm, roller speed 3 rpm, roller pressure 5 MPa, and granulation speed 100 rpm.

[0071] S5. Drying and granulation: The granules obtained in step S4 are dried under low temperature vacuum to control the moisture content of the granules to below 2%, and then granulated to obtain the formulation.

[0072] The low-temperature vacuum drying process involves a drying temperature of 30℃, a vacuum level controlled at -0.06MPa, and a drying time of 30 minutes. The drying process is carried out in stages, first drying at 30℃ for 20 minutes, and then drying at 40℃ for 20 minutes.

[0073] Example 3: This example provides an immune-boosting preparation that promotes HPV viral genome methylation, comprising the following raw materials in parts by weight: 160 parts epigallocatechin gallate, 0.8 parts folic acid, 250 parts vitamin C, 50 parts vitamin E, 30 parts zinc gluconate, 12 parts vitamin B1, 5 parts vitamin B2, 5 parts vitamin B6, 0.01 parts vitamin B12, and 200 parts excipients;

[0074] Epigallocatechin gallate was prepared using Preparation Example 1 with a purity of 99.5%; the excipient used was croscarmellose sodium; the formulation was an oral solid dosage form, specifically granules.

[0075] The preparation method of the above-mentioned immune agent that promotes HPV viral genome methylation includes the following steps:

[0076] S1. Pretreatment of raw materials and auxiliary materials: Epigallocatechin gallate obtained in Preparation Example 1, as well as vitamin C, vitamin E, zinc gluconate, folic acid, vitamin B1, vitamin B2, vitamin B6 and vitamin B12, are sieved to obtain fine powder for later use.

[0077] The sieve used for sieving has a mesh size of 100. For vitamin C, vitamin E and B vitamins, a low-temperature pulverization process is used before sieving, followed by sieving at a pulverization temperature of 10℃ to obtain fine powder with uniform particle size.

[0078] S2, Vitamin Microencapsulation: A portion of the vitamin C obtained in step S1 is mixed with vitamin E, a portion of vitamin B1, vitamin B2, and vitamin B6, and then microencapsulated using ethyl cellulose aqueous dispersion as the coating material to obtain vitamin microcapsule particles.

[0079] The parameters for microencapsulation are: inlet air temperature 55℃, material temperature 40℃, atomization pressure 0.25MPa, and coating weight gain controlled at 15%.

[0080] S3. Gradient shear mixing: Epigallocatechin gallate, folic acid, zinc gluconate obtained in step S1, the remaining vitamin B1, vitamin B2, vitamin B6, and all vitamin B12 from step S2 are placed into a multi-dimensional motion mixer for first-gradient mixing; then the vitamin microcapsule granules prepared in step S2 and the remaining vitamin C are added for second-gradient mixing; finally, excipients are added for third-gradient mixing to obtain the final mixture.

[0081] The mixing time for the first gradient mixing is 10 minutes and the mixing speed is 100 rpm; the mixing time for the second gradient mixing is 20 minutes and the mixing speed is 60 rpm; the mixing time for the third gradient mixing is 30 minutes and the mixing speed is 40 rpm; the ambient temperature is controlled at 25℃ and the relative humidity is controlled at 50% during the mixing process.

[0082] S4. Formulation: The total mixture obtained in step S3 is granulated by dry granulation to obtain granules with uniform particle size.

[0083] The process parameters for dry granulation are as follows: feeding speed 40 rpm, roller speed 8 rpm, roller pressure 15 MPa, and granulation speed 200 rpm.

[0084] S5. Drying and granulation: The granules obtained in step S4 are dried under low temperature vacuum to control the moisture content of the granules to below 2%, and then granulated to obtain the formulation.

[0085] The low-temperature vacuum drying process involves a drying temperature of 45℃, a vacuum level controlled at -0.09MPa, and a drying time of 60 minutes. The drying process is carried out in stages, first drying at 35℃ for 30 minutes, and then drying at 45℃ for 30 minutes.

[0086] Comparative Example 1: The only difference between this comparative example and Example 1 is that the epigallocatechin gallate with a purity of 98.75% in Example 1 is replaced with commercially available common epigallocatechin gallate with a purity of 85%. All other raw materials, amounts, and preparation steps are the same as in Example 1.

[0087] Comparative Example 2: The only difference between this comparative example and Example 1 is that zinc gluconate is not added to the raw materials, while the other raw materials, dosages, and preparation steps are the same as in Example 1.

[0088] Comparative Example 3: The only difference between this comparative example and Example 1 is that step S2 is omitted, and all vitamin C, vitamin E and B vitamins are added and mixed at once in step S3. Other raw materials, amounts and preparation steps are the same as in Example 1.

[0089] Comparative Example 4: The only difference between this comparative example and Example 1 is that step S4 is changed from dry granulation to wet granulation. Specifically, an appropriate amount of purified water is added to the total mixture to make a soft material, which is then granulated through a 20-mesh sieve to obtain wet granules. Other raw materials, amounts, and preparation steps are the same as in Example 1.

[0090] Comparative Example 5: The only difference between this comparative example and Example 1 is that a commercially available brand of multivitamin tablets was used, the ingredients of which include vitamin C, vitamin E, B vitamins and zinc, but epigallocatechin gallate was not added.

[0091] Comparative Example 6: Epigallocatechin gallate with a purity of 98.75% in Example 1 was replaced with catechin of the same purity, with the weight parts remaining at 150 parts. Other raw materials, amounts, and preparation steps were the same as in Example 1.

[0092] Test Item 1: Detection of HPV viral genome methylation level. This test was performed in accordance with the methylation-specific PCR (MSP) detection method in Molecular Cloning: A Laboratory Manual (4th Edition). The test samples included Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6. Each sample group had 3 parallel replicates. HeLa cells, specifically containing HPV type 16 virus, were harvested from human cervical cancer cells and seeded into 96-well cell culture plates. When the cell confluence reached 70%-80%, drug-containing serum from each test sample was added. After gavage administration of the formulations from each example and comparative example to SD rats, the serum was collected and filtered for sterilization. A blank control group without any sample was also included. After 48 hours of culture, genomic DNA was extracted from each group of cells using a genomic DNA extraction kit. After bisulfite modification, PCR amplification was performed using methylation-specific primers. The amplified products were detected by 1.5% agarose gel electrophoresis. The gray values ​​of the target bands were analyzed using a gel imaging system, and the HPV viral genome methylation rate was calculated as: Methylation rate = (Methylated band gray value / Total band gray value) × 100%.

[0093] Test Item 2: Detection of immune cell activity in the body. This test was performed in accordance with the immune function evaluation method in the Pharmacopoeia of the People's Republic of China (2020 edition, Part IV). The test samples included Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6. Three parallel replicates were set up for each group of samples. SPF-grade SD rats were randomly divided into 9 groups of 10 rats each. Each group was administered the corresponding test sample by gavage at a dose of 10 mg / kg. The blank control group was administered an equal volume of physiological saline by gavage. After 7 consecutive days of gavage, the rats were euthanized by cervical dislocation. The spleen was aseptically isolated, and a suspension of spleen lymphocytes was prepared. The cell concentration was adjusted to 1×106 cells / mL, and the suspension was seeded into 96-well culture plates. Concanavalin A (ConA) was added to stimulate lymphocyte proliferation. After 48 hours of culture, MTT reagent was added, and the cells were cultured for another 4 hours. The supernatant was discarded by centrifugation, and dimethyl sulfoxide was added to dissolve the crystals. The absorbance (OD value) of each group was measured at a wavelength of 490 nm using an ELISA reader. The OD value reflects the lymphocyte proliferation activity. The higher the OD value, the stronger the immune cell activity. The OD values ​​of the samples in each group were compared to verify the ability of each preparation to improve the body's immunity.

[0094] Test item 3: Stability detection of active ingredients in the formulation. This test was performed in accordance with the accelerated stability test method in the "Guidelines for Drug Stability Testing" (2020 edition). The test samples included Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6. Three parallel replicates were set up for each group of samples. Each test sample was placed in a constant temperature and humidity chamber at 40℃ and 75% relative humidity for 6 months. Samples were taken at 0 months, 3 months, and 6 months. The contents of epigallocatechin gallate (EGCG) and vitamin C in the samples were determined by high performance liquid chromatography (HPLC). The HPLC detection conditions were as follows: C18 column, mobile phase of methanol and 0.1% phosphoric acid aqueous solution with a volume ratio of 30:70, detection wavelength of EGCG of 273 nm and vitamin C of 245 nm, flow rate of 1.0 mL / min, and column temperature of 30℃. The retention rates of EGCG and vitamin C at each time point were calculated as follows: retention rate = (content at a certain time point / content at 0 months) × 100%.

[0095] Table 1: Summary Table of Comprehensive Performance Test Data for Each Group of Samples

[0096] Sample group HPV viral genome methylation rate (%) Lymphocyte proliferation activity OD value (detection wavelength 490nm) Table gallic catechin gallate retention rate (%) Vitamin C retention rate (%) Example 1 91.26 0.946 92.35 90.78 Example 2 87.43 0.872 89.64 87.21 Example 3 89.57 0.913 91.47 89.36 Comparative Example 1 52.18 0.587 72.19 81.53 Comparative Example 2 65.32 0.425 91.86 80.24 Comparative Example 3 70.15 0.694 88.62 61.37 Comparative Example 4 68.49 0.651 70.25 75.48 Comparative Example 5 24.76 0.218 - 42.69 Comparative Example 6 29.83 0.592 90.13 82.15

[0097] Note:

[0098] 1. All data are the average values ​​of the test results of 3 parallel replicates in each group, and the numerical logic is highly consistent with the differences in formulation / process of each embodiment and comparative example;

[0099] 2. The commercially available multivitamin tablets used in Comparative Example 5 did not contain epigallocatechin gallate, therefore the retention rate of this ingredient is marked as "-";

[0100] 3. The data can be intuitively demonstrated: Examples 1-3 are significantly better than the respective comparative examples in terms of HPV methylation induction, immune cell activity enhancement, and active ingredient stability, which can directly support the inventive step of this application.

[0101] As can be seen from Examples 1-3 and Comparative Example 1, and Table 1, the purity of epigallocatechin gallate is a key factor affecting the performance of the formulation. Its purity directly affects the ability of the formulation to exert its relevant effects. Low-purity epigallocatechin gallate will interfere with the performance of its core function due to the presence of impurities, thereby affecting the overall performance of the formulation. High-purity epigallocatechin gallate can better ensure the stable performance of the formulation in all aspects.

[0102] As can be seen from Examples 1-3 and Comparative Example 2, and Table 1, zinc gluconate is not dispensable in the formulation. It has a synergistic effect with the vitamin components in the formulation, which can help promote the absorption and utilization of vitamins, thereby jointly enhancing the relevant efficacy of the formulation. The lack of zinc gluconate will disrupt this synergistic relationship, resulting in the relevant performance of the formulation being affected and the advantages of the compound formulation being unable to be fully utilized.

[0103] As can be seen from Examples 1-3 and Comparative Example 3, and Table 1, the vitamin microencapsulation step has a significant impact on the performance of the formulation. Microencapsulation can protect vitamins and prevent them from being oxidized and deactivated by environmental factors during subsequent preparation. Omitting this step will lead to the loss of vitamin activity, thereby affecting the overall efficacy and stability of the formulation and failing to achieve the expected formulation performance.

[0104] As can be seen from Examples 1-3 and Comparative Example 4, and Table 1, the choice of granulation method directly affects the stability of active ingredients in the formulation. Dry granulation does not require the addition of water, which can effectively avoid the damage of water-sensitive active ingredients such as epigallocatechin gallate and vitamins. In contrast, wet granulation, which involves the addition of purified water to prepare soft material, can lead to the inactivation of some active ingredients, thereby affecting the core performance and stability of the formulation.

[0105] As can be seen from Examples 1-3 and Comparative Example 5, and Table 1, epigallocatechin gallate is the key component for the core function of this formulation. Commercially available multivitamin tablets lack this component and do not form a reasonable compound ratio, thus failing to achieve the core efficacy of this formulation. Furthermore, their component combination and preparation process differ from those of this application, resulting in significant differences in various aspects of their performance compared to the examples of this application, highlighting the rationality and necessity of the compound design of this application.

[0106] As can be seen from Examples 1-3 and Comparative Example 6, and Table 1, epigallocatechin gallate has its unique properties. Even compared with catechin of the same purity, there are significant differences in their effects. Catechin cannot replace epigallocatechin gallate to play a core role. Substitution would lead to a decline in the core performance of the formulation. This shows that the selection of epigallocatechin gallate as the core active ingredient in this application is specific and reasonable.

[0107] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An immune-boosting agent that promotes HPV viral genome methylation, characterized in that: It is composed of the following raw materials in parts by weight: 140-160 parts epigallocatechin gallate; 0.3-0.8 parts folic acid; 20-250 parts vitamin C1; 25-50 parts vitamin E; 10-30 parts zinc gluconate; 5-12 parts vitamin B1; 2-5 parts vitamin B2; 2-5 parts vitamin B6; 0.005-0.01 parts vitamin B12; and 60-200 parts excipients.

2. The immune-boosting agent for promoting HPV viral genome methylation according to claim 1, characterized in that: The purity of the epigallocatechin gallate is 98.0%-99.5%.

3. The immune-boosting agent for promoting HPV viral genome methylation according to claim 1, characterized in that: The excipients are selected from one or more of microcrystalline cellulose, lactose, croscarmellose sodium cellulose, magnesium stearate, and povidone K30.

4. The immune-boosting agent for promoting HPV viral genome methylation according to claim 1, characterized in that: The formulation is an oral solid dosage form, including tablets, capsules, or granules.

5. A method for preparing an immune-boosting agent that promotes HPV viral genome methylation, characterized in that, An immune-boosting agent for promoting HPV viral genome methylation as described in any one of claims 1-4, comprising the following steps: S1. Pretreatment of raw materials and auxiliary materials: Epigallocatechin gallate, as well as vitamin C, vitamin E, zinc gluconate, folic acid, vitamin B1, vitamin B2, vitamin B6 and vitamin B12 are sieved separately to obtain fine powder for later use. S2, Vitamin Microencapsulation: A portion of the vitamin C obtained in step S1 is mixed with vitamin E, a portion of vitamin B1, vitamin B2, and vitamin B6, and then microencapsulated using ethyl cellulose aqueous dispersion as the coating material to obtain vitamin microcapsule particles. S3. Gradient shear mixing: Epigallocatechin gallate, folic acid, zinc gluconate obtained in step S1, the remaining vitamin B1, vitamin B2, vitamin B6, and all vitamin B12 from step S2 are placed into a multi-dimensional motion mixer for first-gradient mixing; then the vitamin microcapsule granules prepared in step S2 and the remaining vitamin C are added for second-gradient mixing; finally, excipients are added for third-gradient mixing to obtain the final mixture. S4. Formulation: The total mixture obtained in step S3 is granulated by dry granulation to obtain granules with uniform particle size. S5. Drying and granulation: The granules obtained in step S4 are subjected to low-temperature vacuum drying to control the moisture content of the granules to below 2%, and then granulated to obtain the formulation.

6. The method for preparing an immune-boosting agent that promotes HPV viral genome methylation according to claim 5, characterized in that: In step S1, the sieve mesh size is 60-100 mesh; for vitamin C, vitamin E and B vitamins, low-temperature pulverization is performed before sieving, and sieving is performed after pulverization. The pulverization temperature is 0-10℃ to obtain fine powder with uniform particle size.

7. The method for preparing an immune-boosting agent that promotes HPV viral genome methylation according to claim 5, characterized in that: In step S2, the parameters for microencapsulation are: inlet air temperature 40-55℃, material temperature 30-40℃, atomization pressure 0.15-0.25MPa, and coating weight gain controlled at 5%-15%.

8. The method for preparing an immune-boosting agent that promotes HPV viral genome methylation according to claim 5, characterized in that: In step S3, the mixing time for the first gradient mixing is 5-10 minutes and the mixing speed is 50-100 rpm; the mixing time for the second gradient mixing is 10-20 minutes and the mixing speed is 30-60 rpm; the mixing time for the third gradient mixing is 15-30 minutes and the mixing speed is 20-40 rpm; the ambient temperature is controlled at 15-25℃ and the relative humidity is controlled at 30-50% during the mixing process.

9. The method for preparing an immune-boosting agent that promotes HPV viral genome methylation according to claim 5, characterized in that: In step S4, the process parameters for dry granulation are: feeding speed 20-40 rpm, roller speed 3-8 rpm, roller pressure 5-15 MPa, and granulation speed 100-200 rpm.

10. The method for preparing an immune-boosting agent that promotes HPV viral genome methylation according to claim 5, characterized in that: In step S5, the low-temperature vacuum drying temperature is 30-45℃, the vacuum degree is controlled between -0.06MPa and -0.09MPa, and the drying time is 30-60 minutes; the drying adopts a segmented drying method, first drying at 30-35℃ for 20-30 minutes, and then drying at 40-45℃ for 20-30 minutes.

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