Cell activity regeneration compound and preparation method thereof

Abstract

The invention discloses a cell activity regeneration compound and a preparation method thereof, and belongs to the technical field of cell regeneration. The composition is composed of resveratrol, cycloastragenol, recombinant humanized collagen, L-arginine, citrulline, lysine, ginsenoside, sodium hyaluronate and other components, and has multiple effects of activating cell regeneration, enhancing vaginal wall elasticity, regulating endocrine, preventing premature ovarian failure and the like. The preparation method comprises the steps of dissolving, mixing, homogenizing, filtering, filling, sterilizing and the like, is simple in process, controllable in cost and easy for large-scale production, and has remarkable market competitiveness. Experimental results show that the compound can significantly improve the problems of relaxation, dryness, atrophy and the like of the reproductive system, regulate the sex hormone level and enhance the health of the reproductive system, is suitable for women of all ages, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell regeneration, and relates to a cell viability regeneration complex, and also relates to a preparation method of the complex. Background Art

[0002] The health of the female reproductive system has always been an important topic in the fields of medicine and nursing. With the increase in age, the number of pregnancies, and the change in lifestyle, the female reproductive system faces many problems, such as vaginal relaxation, dryness, atrophy, and sexual function decline. These problems not only affect the quality of life of women, but also may lead to infections and inflammations of the reproductive system.

[0003] Currently, the products for female reproductive system care on the market mainly focus on local lubrication, antibacterial and anti-inflammatory aspects, etc., but most of these products can only solve short-term problems and cannot fundamentally improve the health of the reproductive system. The main deficiencies are as follows: Limited local care effect: The existing female intimate care products mainly consist of lubricants and antibacterial agents. Although they can relieve dryness and itching to a certain extent, they cannot fundamentally improve the elasticity and tightness of the vaginal wall. These products usually lack the activation effect on the cell viability of the reproductive system and are difficult to achieve long-term repair and regeneration.

[0004] Lack of comprehensive conditioning function: Most of the existing products only improve a single problem, such as lubrication or antibacterial, and do not comprehensively consider the overall health of the female reproductive system. For example, they lack the regulatory effect on endocrine and cannot fundamentally solve the reproductive system problems caused by changes in hormone levels.

[0005] Safety issues: Some care products contain chemical preservatives or irritating ingredients, which may cause secondary harm to the reproductive system, especially for women with sensitive constitutions. In addition, some products may interfere with the normal physiological functions of the reproductive system and even affect fertility.

[0006] Unable to activate cell regeneration: There is a lack of a complex in the prior art that can effectively activate the cell regeneration of the reproductive system. Most products can only provide short-term soothing effects and cannot promote cell viability and the synthesis of elastin, and are difficult to fundamentally improve the relaxation and atrophy problems of the reproductive system.

[0007] In summary, there are still many problems with the existing cell viability regeneration drugs and skin care products in terms of effect, comprehensive conditioning, safety, etc. Therefore, it is of great significance to develop a complex that can effectively promote cell viability regeneration and is easy to use, safe and without side effects. Summary of the Invention

[0008] The object of the present invention is to provide a cell viability regeneration complex and a preparation method thereof, aiming to activate the regeneration ability of cells in the female reproductive system through the synergistic effect of various bioactive components, enhance the elasticity and firmness of the vaginal wall, regulate the endocrine system at the same time, prevent premature ovarian failure, and fundamentally improve the health of the female reproductive system.

[0009] The first technical solution adopted by the present invention is a cell viability regeneration complex, which is composed of the following components by mass percentage: resveratrol 0.1 - 0.8%, cycloastragenol 0.1 - 1%, recombinant humanized type I collagen 0.1 - 0.5%, recombinant humanized type III collagen 0.1 - 0.5%, L-arginine 0.1 - 1%, citrulline 0.1 - 1%, lysine 0.1 - 1%, ginsenoside 0.1 - 0.5%, sodium hyaluronate 0.1 - 1%, and the rest is purified water.

[0010] The characteristics of this technical solution also lie in: A cell viability regeneration complex, which is composed of the following components by mass percentage: resveratrol 0.4%, cycloastragenol 0.5%, recombinant humanized type I collagen 0.3%, recombinant humanized type III collagen 0.3%, L-arginine 0.5%, citrulline 0.5%, lysine 0.5%, ginsenoside 0.3%, sodium hyaluronate 0.5%, and the rest is purified water.

[0011] The second technical solution adopted by the present invention is a preparation method of a cell viability regeneration complex, which is specifically implemented according to the following steps: S1. Raw material preparation: Weigh each component according to the above mass percentage to ensure that the purity and quality of each component meet the requirements; S2. Dissolution: Dissolve resveratrol, cycloastragenol, ginsenoside, and sodium hyaluronate in purified water respectively, and stir until completely dissolved; S3. Mixing: Mix the dissolved resveratrol, cycloastragenol, ginsenoside, and sodium hyaluronate solutions, and stir evenly; S4. Add collagen: Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixed solution, and stir until completely dissolved; S5. Add amino acids: Add L-arginine, citrulline, and lysine to the mixed solution, and stir until completely dissolved; S6. Homogenization: Homogenize the mixed solution to ensure that each component is evenly distributed; S7. Filtration: Filter the homogenized mixture through a microporous membrane to remove impurities and obtain a clear complex solution; S8. Filling: Fill the filtered complex solution into a sterile container and seal it for storage; S9. Sterilization: Perform low-temperature sterilization on the filled complex to finally obtain a cell viability regeneration complex.

[0012] The features of this technical solution also lie in: In step S1, for the components of plant origin, dry them at 50 - 70 °C for 6 - 10 hours and crush them to a particle size of < 40 mesh.

[0013] The dissolution temperature in S2 is 25 - 40 °C. Use a magnetic stirrer or mechanical stirrer with a stirring speed of 200 - 400 rpm.

[0014] In S3, the mixing temperature is 25 - 40 °C; use a paddle stirrer with a stirring speed of 300 - 500 rpm.

[0015] In S4, the dissolution temperature is 25 - 40 °C; the stirring speed is 100 - 200 rpm, and the dissolution time is 1 - 2 hours.

[0016] In S5, the dissolution temperature is 25 - 40 °C, the stirring speed is 200 - 300 rpm, and the dissolution time is 10 - 20 minutes.

[0017] In S6, the homogenization pressure is 50 - 100 MPa, and use a high-pressure homogenizer to circulate and process 3 - 5 times; the processing temperature is 25 - 40 °C, which is controlled by jacket cooling.

[0018] In S7, filter through a 0.22 - 0.25 μm microporous membrane, and the filtration temperature is 25 - 40 °C; the filtration pressure is 0.1 - 0.3 MPa, and use nitrogen to pressurize the filtration.

[0019] In S7, the filter membrane material is a hydrophilic polyethersulfone PES membrane.

[0020] In S8, the filling speed is 50 - 100 mL / min, and use a peristaltic pump for quantitative filling; the container material is a brown glass bottle or a medical-grade polypropylene bottle.

[0021] The cell viability regeneration complex of the present invention and its preparation method have the following technical effects: Comprehensive conditioning and repair: Through the synergistic effect of various bioactive components, it can effectively supplement the nutrients required by the reproductive system and enhance the elasticity and firmness of the vaginal wall. Compared with existing products, this complex can not only provide short-term lubrication effects, but also achieve long-term repair and regeneration by activating the cell regeneration ability.

[0022] Improve the quality of life: This complex can significantly improve problems such as dryness, relaxation, and atrophy of the female reproductive system, and enhance the quality of sexual life. At the same time, its comprehensive conditioning function can regulate the endocrine system, prevent premature ovarian failure, promote smooth, delicate, and rosy skin, delay aging, and thus comprehensively improve the quality of life of women.

[0023] Safety and compatibility: It uses natural plant extracts and recombinant humanized collagen and other ingredients, without chemical preservatives and irritating ingredients, and has good biocompatibility and safety. Recombinant collagen is homologous to the human body, avoiding immune rejection; plant extracts (such as cyclocarya palida alcohol) have low irritation after purification. Compared with existing products, this complex will not cause secondary damage to the reproductive system. Wide range of applicable populations: It is not only suitable for postpartum repair, but also can be used for dryness during menopause, postoperative mucosal repair, and daily maintenance, and is suitable for women of all ages for long-term use.

[0024] Market prospect: It realizes the synergistic effect of cell vitality regeneration, tissue structure repair, and endocrine balance, filling the functional gap of existing technologies. At the same time, the preparation process of this complex is simple, the cost is controllable, it is easy to scale production, and it has significant market competitiveness. Description of the drawings

[0025] Figure 1 It is a flow chart of the preparation method of the cell vitality regeneration complex of the present invention. Detailed implementation manners

[0026] The present invention will be described in detail below in conjunction with the drawings and specific implementation manners.

[0027] A cell vitality regeneration complex of the present invention is composed of a variety of bioactive ingredients, and the components are composed by mass percentage: resveratrol 0.1 - 0.8%, cyclocarya palida alcohol 0.1 - 1%, recombinant humanized type I collagen 0.1 - 0.5%, recombinant humanized type III collagen 0.1 - 0.5%, L-arginine 0.1 - 1%, citrulline 0.1 - 1%, lysine 0.1 - 1%, ginsenoside 0.1 - 0.5%, sodium hyaluronate 0.1 - 1%, and the rest is purified water.

[0028] Through scientific compatibility, such as resveratrol, cyclocarya palida alcohol, recombinant collagen, amino acids, ginsenoside, and sodium hyaluronate. They work together synergistically to achieve cell vitality regeneration and reproductive system repair, specifically as follows: Resveratrol (0.1 - 0.8%): As a polyphenolic compound, it enhances the antioxidant capacity of cells by activating the SIRT1 pathway, scavenges ROS (reactive oxygen species), and inhibits the NF-κB-mediated inflammatory response.

[0029] Cycloastragenol (0.1 - 1%): A triterpenoid saponin component that promotes the expression of endogenous antioxidant enzymes (such as SOD, GSH-Px) by upregulating the Nrf2 pathway, and forms an "exogenous + endogenous" dual-effect antioxidant network with resveratrol.

[0030] Resveratrol has antioxidant, anti-inflammatory, and cytoprotective effects, capable of scavenging free radicals and protecting cells from oxidative damage. Cycloastragenol, on the other hand, has the effect of promoting cell regeneration and repair. The two work synergistically to effectively activate the regenerative capacity of cells and promote the vitality of reproductive system cells.

[0031] Ginsenoside (0.1 - 0.5%): Ginsenoside has the effects of regulating the endocrine system and enhancing immunity, capable of activating ovarian function and preventing premature ovarian failure. By inhibiting the activities of COX-2 and iNOS, it reduces the release of inflammatory factors such as prostaglandin E2 (PGE2) and nitric oxide (NO), and synergistically reduces oxidative stress damage.

[0032] Recombinant humanized type I / III collagen (each 0.1 - 0.5%): Type I collagen: Accounts for more than 80% of the dermis of the skin, provides mechanical strength, and directly supplements the collagen fibers lost due to aging.

[0033] Type III collagen: Promotes the migration and proliferation of fibroblasts and accelerates wound repair.

[0034] Recombinant humanized type I and type III collagen are important components of the extracellular matrix, capable of enhancing cell-cell connections and tissue elasticity. By supplementing collagen, the elasticity and firmness of the vaginal wall can be significantly improved, reducing relaxation and atrophy.

[0035] Advantages of recombinant technology: Humanized collagen expressed through genetic engineering has a homology of >95% with the human collagen sequence, avoiding the immunogenic risk of animal-derived collagen.

[0036] L-Arginine, citrulline, lysine (each 0.1 - 1%): L-Arginine, citrulline, and lysine are important raw materials for cell metabolism and protein synthesis, capable of promoting cell growth and repair.

[0037] L-Arginine: As a precursor of nitric oxide (NO), it dilates microvessels through the NO-cGMP pathway, improves local blood oxygen supply, and promotes collagen synthesis.

[0038] Lysine: A cofactor of hydroxylase, directly participates in collagen cross-linking, and stabilizes the three-dimensional structure of collagen fibers.

[0039] Citrulline: Reduces ammonia toxicity through the urea cycle, indirectly protects mitochondrial function, and maintains the efficiency of cell anabolism.

[0040] Sodium hyaluronate (0.1 - 1%): Sodium hyaluronate has good moisturizing properties, can maintain the moist environment of the reproductive system, reduce dryness and itching. At the same time, sodium hyaluronate can also promote the absorption and utilization of cells and enhance the effects of other ingredients.

[0041] Through molecular weight fractionation (such as low molecular weight HA penetrating into the dermis layer and high molecular weight HA forming a water-locking film on the epidermis), the dual functions of "deep moisturizing + surface protection" are achieved.

[0042] Synergistically with collagen to enhance the hydration of the extracellular matrix (ECM) and increase the skin elastic modulus by 20 - 30%.

[0043] Multiple components synergistically act through multiple pathways such as antioxidant, anti-inflammatory, collagen synthesis, microcirculation improvement, and moisturizing repair, breaking through the limitations of the effects of single components and comprehensively enhancing the efficiency of cell vitality regeneration.

[0044] Replacement of chemically synthesized components with natural plant extracts: Resveratrol and cyclocophorol: Extracted from grapes and astragalus respectively, with definite safety and low irritation (confirmed by EC50 test, the half inhibitory concentration for HaCaT cells > 500 μM).

[0045] Ginsenoside: Using supercritical CO2 extraction technology to avoid organic solvent residues and ensure a purity > 98%.

[0046] Safety advantages of bioengineered collagen: Recombinant humanized collagen is produced through the Pichia pastoris expression system. After detection of host cell protein residues (HCP < 1 ng / mg) and endotoxin control (< 0.1 EU / mg), the risk of virus contamination of animal-derived collagen is avoided.

[0047] Physiological compatibility of amino acids: L-arginine and lysine are essential amino acids for the human body, and citrulline is a natural metabolic intermediate. All three have passed GRAS (Generally Recognized as Safe) certification and have no risk of cumulative toxicity.

[0048] The introduction of natural and bioengineered components significantly reduces the irritation of the product (confirmed by patch test, the sensitization rate < 0.1%), making it suitable for sensitive skin and long-term use scenarios.

[0049] Low-temperature dissolution and mixing control: The dissolution temperature is strictly controlled at 25 - 40 °C to avoid the loss of activity of resveratrol (thermal decomposition temperature > 160 °C but prone to oxidation under the synergistic effect of light and heat) and collagen (denaturation temperature 45 °C).

[0050] During the mixing stage, medium-speed stirring at 300 - 500 rpm is adopted to avoid protein aggregation caused by high-speed shearing and prevent uneven mixing at low speed.

[0051] High-pressure homogenization nanotechnology: Through high-pressure homogenization at 50 - 100 MPa, the particle size of the collagen-hyaluronic acid complex is reduced to <200 nm, forming a stable nanoemulsion (absolute value of Zeta potential > 30 mV), and delaying the precipitation of components caused by Ostwald ripening.

[0052] Nanomization increases the specific surface area of the active ingredients by 5 - 10 times, improving solubility and transdermal rate.

[0053] The complex of the present invention activates the cell regeneration ability and promotes the synthesis of elastin and collagen through the synergistic effect of multiple bioactive ingredients. The specific mechanism is as follows: Antioxidant protection: Resveratrol and cyclocarya pallida alcohol can scavenge free radicals in cells, reduce oxidative stress, and protect cells from oxidative damage. This antioxidant protection provides a good environment for cell regeneration.

[0054] Activation of cell signaling pathways: Ginsenosides can activate multiple signaling pathways in cells, such as the PI3K / Akt pathway and the MAPK pathway, promoting cell proliferation and differentiation. At the same time, recombinant humanized collagen can bind to receptors on the cell surface, activating the synthesis and remodeling of the extracellular matrix.

[0055] Nutrient supplementation and metabolism promotion: Amino acids such as L-arginine, citrulline, and lysine are important raw materials for cell metabolism and protein synthesis, providing necessary nutritional support for cell regeneration. Sodium hyaluronate can maintain the water environment of cells, promoting cell absorption and utilization.

[0056] Endocrine regulation: Ginsenosides and cyclocarya pallida alcohol can regulate female endocrine, activate ovarian function, and promote the secretion of estrogen and progesterone. These hormones can improve the physiological functions of the reproductive system and enhance cell vitality and regeneration ability.

[0057] Low-temperature sterilization and aseptic filling: Pasteurization (60 - 80 °C, 20 - 40 minutes) or irradiation sterilization (5 - 15 kGy) is adopted, reducing the loss of heat-sensitive components by > 50% compared with traditional high-temperature sterilization at 121 °C.

[0058] The filling process is completed in a Class 100 clean environment, filled with nitrogen and sealed (oxygen residue < 0.5%) to inhibit oxidation reactions.

[0059] After accelerated stability testing (40°C / 75%RH, 6 months), the resveratrol retention rate > 90%, and the collagen activity (detecting the integrity of the triple helix structure by CD spectroscopy) was maintained > 85%. The product shelf life was extended to 24 months.

[0060] Raw material level safety control: Plant components such as resveratrol and cyclocophorol need to pass heavy metal (Pb < 1 ppm) and pesticide residue (organophosphorus < 0.01 ppm) detection.

[0061] Recombinant collagen needs to be controlled for host DNA residue detection (< 10 pg / mg) and endotoxin (< 0.1 EU / mg).

[0062] As Figure 1 shown, a cell viability regeneration complex and its preparation method of the present invention are applicable to industrial production, with both high efficiency and economy, providing technical support for the large-scale application of the cell viability regeneration complex. Specifically, it is implemented according to the following steps: S1. Raw material preparation Accurately weigh each component by mass percentage: resveratrol (0.1 - 0.8%), cyclocophorol (0.1 - 1%), recombinant humanized type I collagen (0.1 - 0.5%), recombinant humanized type III collagen (0.1 - 0.5%), L-arginine (0.1 - 1%), citrulline (0.1 - 1%), lysine (0.1 - 1%), ginsenoside (0.1 - 0.5%), sodium hyaluronate (0.1 - 1%), and the balance is purified water.

[0063] High-precision weighing is the basis for ensuring formula consistency and batch-to-batch stability, especially crucial for low-concentration active ingredients (such as resveratrol). Use an analytical balance (accuracy ±0.0001 g) to ensure that the error of each component ≤ ±1%.

[0064] Plant-derived components (such as ginsenoside) need to be dried at 50 - 70°C for 6 - 10 hours and pulverized to a particle size < 40 mesh (aperture about 0.425 mm). Drying removes moisture to prevent microbial growth, and pulverization increases the specific surface area to improve subsequent dissolution efficiency.

[0065] This step is used to ensure raw material purity and activity, avoiding impurity interference in subsequent processes. The pulverized raw materials are more easily dissolved, reducing process time and energy consumption.

[0066] S2. Dissolution Dissolve resveratrol, cyclocophorol, ginsenoside, and sodium hyaluronate in purified water respectively.

[0067] Dissolution temperature: 25 - 40°C, controlled by a constant temperature water bath. Resveratrol and ginsenosides are heat-sensitive components. Temperatures above 40°C are likely to cause oxidation or degradation; temperatures below 25°C will result in a decreased dissolution rate.

[0068] Stirring speed: 200 - 400 rpm, using a magnetic stirrer or mechanical stirrer. Moderate stirring accelerates molecular diffusion and avoids the formation of agglomerates due to excessive local concentration.

[0069] Dissolution time: 30 - 60 minutes until the solution is clear and free of particles. Ensure that the active ingredients are fully dissolved to avoid the influence of undissolved particles on homogenization and filtration effects.

[0070] Through this step, a uniform and transparent solution of active ingredients can be obtained, providing a stable system for subsequent mixing, while reducing the loss of ingredients or product non-uniformity caused by insufficient dissolution.

[0071] S3. Mixing Mix the dissolved resveratrol, cyclocophorol, ginsenosides, and sodium hyaluronate solutions and stir evenly.

[0072] Mixing temperature: 25 - 40°C, maintained the same as the water bath dissolution temperature. Temperature fluctuations may cause some components to precipitate (e.g., sodium hyaluronate is prone to form gels at low temperatures).

[0073] Stirring speed: 300 - 500 rpm, using a paddle stirrer. Higher rotation speeds promote the rapid mixing of multiphase liquids and avoid stratification or precipitation.

[0074] Mixing time: 20 - 40 minutes to ensure that the solutions are fully mixed to form a homogeneous premix.

[0075] After this step, a stable mixture can be formed, with the active ingredients evenly distributed, no visible stratification or precipitation, providing a well-compatible basic system for subsequent addition of collagen and amino acids.

[0076] S4. Add collagen Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixture and stir until completely dissolved.

[0077] Dissolution temperature: 25 - 40°C, to avoid protein denaturation caused by high temperatures. Collagen is prone to structural damage and loss of biological activity at temperatures above 45°C.

[0078] Stirring speed: 100 - 200 rpm, using low-speed stirring. High shear forces may damage the tertiary structure of collagen and reduce its repair efficacy.

[0079] Dissolution time: 1 - 2 hours until the solution becomes homogeneous and viscous. Collagen has a relatively large molecular weight (about 300 kDa) and needs to be hydrated slowly to maintain its native conformation.

[0080] After this step, the collagen is completely dissolved and retains its biological activity, forming a viscoelastic composite solution. Avoid protein aggregation or inactivation caused by too fast stirring or improper temperature.

[0081] S5. Add amino acids Add L-arginine, citrulline, and lysine to the mixture and stir until completely dissolved.

[0082] Dissolution temperature: 25 - 40 °C, the same as the collagen dissolution temperature. Amino acids may undergo the Maillard reaction at high temperatures, generating brown by-products.

[0083] Stirring speed: 200 - 300 rpm, avoid violent stirring to introduce air bubbles. Amino acids are easily soluble in water, and moderate stirring can achieve rapid dispersion.

[0084] Dissolution time: 10 - 20 minutes. Small molecule amino acids dissolve quickly, and prolonging the stirring time is not beneficial and may increase the risk of oxidation.

[0085] After this step, the amino acids are completely dissolved, forming a synergistic system with collagen and active ingredients. At the same time, the generation of air bubbles is reduced, ensuring the clear appearance of the composite solution.

[0086] S6. Homogenization Homogenize the mixture to ensure uniform distribution of each component.

[0087] Homogenization pressure: 50 - 100 MPa, use a high-pressure homogenizer to circulate and process 3 - 5 times. High-pressure shear force can break colloidal particles, reduce the particle size to <200 nm, and improve the stability of the system.

[0088] Processing temperature: 25 - 40 °C, controlled by jacket cooling. Heat is generated during the homogenization process, and temperature control is required to prevent degradation of heat-sensitive components (such as resveratrol).

[0089] Number of cycles: 3 - 5 times, adjusted according to the viscosity of the system. Multiple cycles ensure uniform particle size distribution and avoid unprocessed areas.

[0090] After this step, a nano-scale uniform dispersion system can be formed, significantly improving the transdermal absorption rate and reducing stratification or precipitation during storage.

[0091] S7. Filtration Filter the homogenized mixture through a 0.22 - 0.25 μm microporous membrane to remove impurities.

[0092] Filtration temperature: 25 - 40°C, maintaining the same as the homogenization temperature. A decrease in temperature may cause macromolecules (such as collagen) to precipitate and clog the filter membrane.

[0093] Filtration pressure: 0.1 - 0.3 MPa, using nitrogen gas for pressurized filtration. Low-pressure filtration reduces the adsorption loss of active ingredients and avoids damaging the filter membrane structure under high pressure.

[0094] Filter membrane material: Hydrophilic polyethersulfone PES membrane. The PES membrane has high chemical stability and low protein adsorption rate, suitable for filtering biological preparations.

[0095] Through this step, a sterile and particle-free clarified solution can be obtained, meeting the visible foreign matter standard of the pharmacopoeia. The active ingredients are retained to the maximum extent, and the filtration efficiency > 99.9%.

[0096] S8. Filling Fill the filtered complex solution into a sterile container and seal it for storage.

[0097] Filling environment: Class 100 clean room (ISO 5), laminar air flow velocity 0.45 ± 0.1 m / s. Strictly control microbial and particle contamination to ensure the sterility of the product.

[0098] Filling speed: 50 - 100 mL / min, using a peristaltic pump for quantitative filling. Accurately control the filling volume, with an error ≤ ±1%, to avoid waste or insufficient dosage.

[0099] Container material: Brown glass bottle (light-proof) or medical-grade polypropylene (PP) bottle. Light-proof to prevent degradation of photosensitive ingredients (such as resveratrol), and medical-grade materials ensure compatibility.

[0100] In this step, the filling process is pollution-free and leak-free, and the filling volume per bottle is precisely the same. The container has good sealing performance, and there is no oxidation or moisture loss during long-term storage.

[0101] S9. Sterilization Perform low-temperature sterilization on the filled complex to finally obtain the cell vitality regeneration complex.

[0102] The sterilization method can be as follows: Pasteurization: Maintain at 60 - 80°C for 20 - 40 minutes, suitable for components with good thermal stability.

[0103] Irradiation sterilization: Dose 5 - 15 kGy, using γ-rays or electron beams, suitable for extremely heat-sensitive components.

[0104] Sterilization verification: Biological indicator (such as Bacillus stearothermophilus) challenge test to ensure the sterilization effect (SAL ≤ 10^-6).

[0105] Low-temperature sterilization minimizes the loss of active ingredients while killing microorganisms. Through this step, the sterility assurance level (SAL) of the product meets the pharmacopoeia requirements (≤10^-6). The retention rate of active ingredients is ≥95%, avoiding degradation or inactivation caused by high temperature.

[0106] The preparation method of the present invention optimizes the parameters of each step to ensure the stability and effectiveness of the complex. The specific optimization measures are as follows: Dissolution and mixing: By controlling the dissolution temperature and stirring speed, ensure the complete dissolution and uniform mixing of each component. Using a magnetic stirrer or mechanical stirring can improve the dissolution efficiency and reduce the generation of impurities.

[0107] Homogenization treatment: Use a high-pressure homogenizer for homogenization treatment to ensure the uniform distribution of each component. By controlling the treatment temperature through jacket cooling, the destruction of bioactive ingredients caused by high temperature can be avoided.

[0108] Filtration and sterilization: Remove impurities through microporous membrane filtration to ensure the clarity of the complex. Using low-temperature sterilization treatment can effectively kill bacteria and viruses while maintaining the stability of bioactive ingredients.

[0109] Before using the complex of the present invention, it is recommended to first wash the vulva with warm water to keep the reproductive system clean. Avoid using cleaning products containing fragrances, pigments or irritating ingredients. Take out an appropriate amount of the product (about 0.5 - 1.0 mL), and the dosage can be adjusted according to personal needs.

[0110] Application method: External application: Evenly apply the complex on the vulva, focusing on applying around the vaginal orifice, labia minora and other parts, and gently massage until completely absorbed.

[0111] Internal application: If deeper nourishment and repair are needed, a matching tampon or a special applicator (such as a disposable drug delivery device) can be used to gently send the complex into the vagina to a depth of about 2 - 3 cm, and gently massage to promote absorption.

[0112] Frequency of use: Initial use: It is recommended to use it once every night for 4 - 8 weeks to achieve the best effect.

[0113] Maintenance use: After completing the initial course of treatment, it can be adjusted to 2 - 3 times a week according to personal needs for long-term maintenance of reproductive system health.

[0114] To further illustrate the technical solution of the present invention, the following embodiments are provided: Example 1 S1. Raw material preparation: Weigh each component according to the following mass percentages to ensure that the purity and quality of each component meet the requirements. Specifically, resveratrol 0.1%, cyclocophorol 0.1%, recombinant humanized type I collagen 0.1%, recombinant humanized type III collagen 0.1%, L-arginine 0.1%, citrulline 0.1%, lysine 0.1%, ginsenoside 0.1%, sodium hyaluronate 0.1%, and the rest is purified water.

[0115] Among the above components, for the components of plant origin, dry them at 70 °C for 6 hours and crush them to a particle size of 30 mesh.

[0116] S2. Dissolution: Dissolve resveratrol, cyclocophorol, ginsenoside, and sodium hyaluronate in purified water respectively, and stir until completely dissolved; the dissolution temperature is 25 °C, use a magnetic stirrer, and the stirring speed is 200 rpm.

[0117] S3. Mixing: Mix the dissolved resveratrol, cyclocophorol, ginsenoside, and sodium hyaluronate solutions and stir evenly; the mixing temperature is 25 °C; use a paddle stirrer, and the stirring speed is 300 rpm.

[0118] S4. Add collagen: Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixed solution and stir until completely dissolved; the dissolution temperature is 25 °C; the stirring speed is 100 rpm, and the dissolution time is 1 hour.

[0119] S5. Add amino acids: Add L-arginine, citrulline, and lysine to the mixed solution and stir until completely dissolved; the dissolution temperature is 25 °C, the stirring speed is 200 rpm, and the dissolution time is 10 minutes.

[0120] S6. Homogenization: Homogenize the mixed solution to ensure that each component is evenly distributed; the homogenization pressure is 50 MPa, use a high-pressure homogenizer to circulate and process 3 times; the processing temperature is 25 °C, and it is controlled by jacket cooling.

[0121] S7. Filtration: Filter the homogenized mixed solution through a microporous membrane to remove impurities and obtain a clear complex solution; filter through a 0.22 μm microporous membrane, the filtration temperature is 25 °C; the filtration pressure is 0.1 MPa, and nitrogen is used for pressurized filtration. The membrane material is a hydrophilic polyethersulfone PES membrane.

[0122] S8. Filling: Fill the filtered complex solution into a sterile container and seal it for storage; the filling speed is 50 mL / min, and a peristaltic pump is used for quantitative filling; the container material is a brown glass bottle.

[0123] S9. Sterilization: Perform low-temperature sterilization on the filled complex to finally obtain the cell viability regeneration complex.

[0124] Example 2 S1. Raw material preparation: Weigh each component according to the following mass percentages to ensure that the purity and quality of each component meet the requirements. Specifically, resveratrol is 0.4%, cyclocarya palida alcohol is 0.5%, recombinant humanized type I collagen is 0.3%, recombinant humanized type III collagen is 0.3%, L-arginine is 0.5%, citrulline is 0.5%, lysine is 0.5%, ginsenoside is 0.3%, sodium hyaluronate is 0.5%, and the rest is purified water.

[0125] Among the above components, for the plant-derived components, dry them at 60 °C for 8 hours and crush them to a particle size of 35 mesh.

[0126] S2. Dissolution: Dissolve resveratrol, cyclocarya palida alcohol, ginsenoside, and sodium hyaluronate in purified water respectively, and stir until completely dissolved; the dissolution temperature is 35 °C, mechanical stirring is used, and the stirring speed is 300 rpm.

[0127] S3. Mixing: Mix the dissolved resveratrol, cyclocarya palida alcohol, ginsenoside, and sodium hyaluronate solutions and stir evenly; the mixing temperature is 35 °C; use a paddle stirrer, and the stirring speed is 400 rpm.

[0128] S4. Add collagen: Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixed solution and stir until completely dissolved; the dissolution temperature is 35 °C; the stirring speed is 150 rpm, and the dissolution time is 1.5 hours.

[0129] S5. Add amino acids: Add L-arginine, citrulline, and lysine to the mixed solution and stir until completely dissolved; the dissolution temperature is 35 °C, the stirring speed is 250 rpm, and the dissolution time is 15 minutes.

[0130] S6. Homogenization: Perform homogenization on the mixed solution to ensure that each component is evenly distributed; the homogenization pressure is 75 MPa, and use a high-pressure homogenizer to circulate and process 4 times; the processing temperature is 35 °C, which is controlled by jacket cooling.

[0131] S7. Filtration: Filter the homogenized mixture through a microporous membrane to remove impurities and obtain a clear complex solution; filter through a 0.23 μm microporous membrane at a filtration temperature of 35 °C; the filtration pressure is 0.2 MPa, and nitrogen is used for pressurized filtration. The membrane material is a hydrophilic polyethersulfone PES membrane.

[0132] S8. Filling: Fill the filtered complex solution into a sterile container and seal it for storage; the filling speed is 75 mL / min, and a peristaltic pump is used for quantitative filling; the container material is a medical-grade polypropylene bottle.

[0133] S9. Sterilization: Perform low-temperature sterilization on the filled complex to finally obtain a cell viability regeneration complex.

[0134] Example 3 S1. Raw material preparation: Weigh each component according to the following mass percentages to ensure that the purity and quality of each component meet the requirements. Specifically, resveratrol 0.8%, cyclocarya palynoside 1%, recombinant humanized type I collagen 0.5%, recombinant humanized type III collagen 0.5%, L-arginine 1%, citrulline 1%, lysine 1%, ginsenoside 0.5%, sodium hyaluronate 1%, and the rest is purified water.

[0135] Among the above components, the plant-derived components are dried at 70 °C for 6 hours and pulverized to a particle size of 40 mesh.

[0136] S2. Dissolution: Dissolve resveratrol, cyclocarya palynoside, ginsenoside, and sodium hyaluronate in purified water respectively and stir until completely dissolved; the dissolution temperature is 40 °C, mechanical stirring is used, and the stirring speed is 400 rpm.

[0137] S3. Mixing: Mix the dissolved resveratrol, cyclocarya palynoside, ginsenoside, and sodium hyaluronate solutions and stir evenly; the mixing temperature is 40 °C; a paddle stirrer is used, and the stirring speed is 500 rpm.

[0138] S4. Add collagen: Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixed solution and stir until completely dissolved; the dissolution temperature is 40 °C; the stirring speed is 200 rpm, and the dissolution time is 1 hour.

[0139] S5. Add amino acids: Add L-arginine, citrulline, and lysine to the mixed solution and stir until completely dissolved; the dissolution temperature is 40 °C, the stirring speed is 300 rpm, and the dissolution time is 10 minutes.

[0140] S6. Homogenization: The mixed solution is subjected to homogenization treatment to ensure the uniform distribution of each component; the homogenization pressure is 100 MPa, and it is cycled through a high-pressure homogenizer 3 times; the treatment temperature is 40 °C, which is controlled by jacket cooling.

[0141] S7. Filtration: The homogenized mixed solution is filtered through a microporous filter membrane to remove impurities, and a clear complex solution is obtained; it is filtered through a 0.25-μm microporous filter membrane, and the filtration temperature is 40 °C; the filtration pressure is 0.3 MPa, and nitrogen is used for pressurized filtration. The filter membrane material is a hydrophilic polyethersulfone PES membrane.

[0142] S8. Filling: The filtered complex solution is filled into a sterile container and sealed for storage; the filling speed is 100 mL / min, and a peristaltic pump is used for quantitative filling; the container material is a medical-grade polypropylene bottle.

[0143] S9. Sterilization: The filled complex is subjected to low-temperature sterilization treatment to finally obtain a cell viability regeneration complex.

[0144] To further verify the technical effects of the cell viability regeneration complex of the present invention, the following tests are specifically carried out: Experiment 1: Cell viability detection Experiment purpose: To detect the effect of the cell viability regeneration complex on the cell viability of the female reproductive system.

[0145] Experiment method: 1. Cell culture: Primary cultured human vaginal epithelial cells and endometrial cells are cultured in DMEM medium containing 10% fetal bovine serum respectively.

[0146] 2. Experimental grouping: The cells are divided into a normal group, a model group (induced injury by adding oxidant), a complex administration group, and a single component administration group.

[0147] 3. Cell viability detection: The CCK-8 method is used to detect cell viability, and the absorbance is measured at 460 nm by an enzyme-labeled instrument.

[0148] Experimental results: Cell viability: The cell viability of the model group was significantly lower than that of the normal group (P < 0.05), indicating that the injury model induced by the oxidant was successful. The cell viability of the complex administration group was significantly higher than that of the model group (P < 0.05), while the effect of the single component administration group was not significant. This indicates that the complex of the present invention can significantly improve cell viability and promote cell regeneration.

[0149] Elastin synthesis: Immunofluorescence was used to detect the synthesis of elastin within cells. The results showed that the fluorescence intensity of elastin within cells in the complex administration group was significantly higher than that in the model group (P < 0.05), indicating that the complex could promote the synthesis of elastin.

[0150] Conclusion: The cell viability regeneration complex of the present invention can significantly improve the viability of cells in the female reproductive system, promote the synthesis of elastin, and has good repair and regeneration effects.

[0151] Experiment 2: Animal experiment Experiment purpose: To verify the improvement effect of the cell viability regeneration complex on the relaxation and atrophy of the female reproductive system.

[0152] Experiment method: 1. Animal model: Female SD rats at 6 weeks of age were selected to induce a model of reproductive system relaxation and atrophy through natural aging and reproduction.

[0153] 2. Experimental grouping: The rats were divided into a normal group, a model group, a complex administration group, and a single-component administration group for each component.

[0154] 3. Administration method: Administration was through vaginal administration once a day.

[0155] 4. Administration cycle: The administration cycle was 4 weeks.

[0156] 5. Detection indicators: Morphological observation of the reproductive system: The morphology and weight changes of the vagina, uterus, and ovaries of the rats were observed through dissection.

[0157] Histopathological detection: Hematoxylin-eosin (H&E) staining was used to observe the histological structure changes of the vaginal wall and endometrium.

[0158] Detection of elastin and collagen contents: The contents of elastin and collagen in the tissues were detected by Masson trichrome staining.

[0159] Detection of serum sex hormone levels: ELISA kits were used to detect the levels of sex hormones such as estradiol (E2) and progesterone (P) in the serum.

[0160] Experimental results: 1. Morphological observation of the reproductive system: In the model group, obvious atrophy and relaxation occurred in the vagina, uterus, and ovaries of the rats, and the weight was significantly lower than that in the normal group (P < 0.05).

[0161] The reproductive system morphology of the rats in the complex administration group was significantly better than that in the model group. The vaginal wall thickness increased, and the weights of the uterus and ovaries were significantly higher than those in the model group (P < 0.05), approaching the normal group level.

[0162] The administration groups of each component monomer had a certain improvement effect on the morphology of the reproductive system, but the effect was not as significant as that of the complex group.

[0163] 2. Histopathological examination: In the model group, obvious cell atrophy, disordered arrangement, and broken elastic fibers were observed in the vaginal wall and endometrium of rats.

[0164] The tissue structure of the complex administration group was close to that of the normal group, with neatly arranged cells, significantly increased elastic fibers and collagen fibers, and good tissue repair.

[0165] The tissue repair effect of each component monomer administration group was limited and did not reach the level of the complex group.

[0166] 3. Detection of elastin and collagen contents: The contents of elastin and collagen in the vaginal wall and uterine tissue of rats in the model group were significantly decreased (P<0.05).

[0167] The contents of elastin and collagen in the complex administration group were significantly higher than those in the model group (P<0.05), approaching the level of the normal group, indicating that the complex could significantly promote the synthesis of elastin and collagen.

[0168] Each component monomer administration group had a certain promoting effect on the contents of elastin and collagen, but the effect was not as significant as that of the complex group.

[0169] 4. Detection of serum sex hormone levels: The levels of serum estradiol (E2) and progesterone (P) in the model group were significantly lower than those in the normal group (P<0.05), indicating the decline of reproductive system function.

[0170] The serum sex hormone levels in the complex administration group were significantly higher than those in the model group (P<0.05), approaching the level of the normal group, indicating that the complex could regulate endocrine and activate ovarian function.

[0171] Each component monomer administration group had a certain promoting effect on sex hormone levels, but the effect was not as significant as that of the complex group.

[0172] Conclusion: Through the synergistic effect of various bioactive components, the cell viability regeneration complex of the present invention significantly improved the relaxation and atrophy of the reproductive system in the animal model, promoted the synthesis of elastin and collagen, regulated the endocrine level, and had good repair and regeneration effects. The experimental results showed that the comprehensive effect of the complex was better than that of each component monomer, with a significant synergistic effect.

[0173] Experiment 3: Human clinical trial Purpose of the experiment: Verify the safety and effectiveness of the cell viability regeneration complex in the human body, and evaluate its actual improvement effect on the health of the female reproductive system.

[0174] Experimental method: 1. Subject selection: Select 100 female volunteers aged between 25 and 50, all of whom have varying degrees of problems such as relaxation, dryness, and atrophy of the reproductive system.

[0175] Exclusion criteria: Pregnant and lactating women, patients with severe gynecological diseases or endocrine disorders, and those allergic to the ingredients of the test product.

[0176] 2. Experimental grouping: Randomly divide the subjects into an experimental group (50) and a control group (50).

[0177] The experimental group uses the cell viability regeneration complex of the present invention, and the control group uses a placebo.

[0178] 3. Usage method: Use once a day for 8 consecutive weeks.

[0179] Conduct a questionnaire survey, gynecological examination, and relevant index detection before and after use.

[0180] 4. Detection indicators: Subjective feeling evaluation: Understand the improvement feelings of the subjects on symptoms such as dryness, relaxation, and itching of the reproductive system through a questionnaire survey.

[0181] Gynecological examination: Evaluate the morphological and functional changes of the reproductive system through a gynecological examination, including the elasticity of the vaginal wall and the characteristics of secretions.

[0182] Laboratory test: Detect the serum sex hormone levels (estradiol, progesterone, etc.) and the number of lactobacilli in vaginal secretions.

[0183] Experimental results: 1. Subjective feeling evaluation: After 8 weeks of use, the symptoms such as dryness, relaxation, and itching of the reproductive system in the experimental group were significantly improved, and the satisfaction rate reached over 85%. In contrast, the improvement in the control group was not obvious, and the satisfaction rate was only about 20%.

[0184] The subjects in the experimental group generally reported that the reproductive system was more lubricated and tightened, and the quality of sexual life was significantly improved.

[0185] 2. Gynecological examination results: The elasticity of the vaginal wall in the experimental group was significantly enhanced, the characteristics of vaginal secretions returned to normal, and the number of lactobacilli increased significantly, indicating that the microecology of the reproductive system was improved.

[0186] There were no significant changes in the gynecological examination indicators of the subjects in the control group.

[0187] 3. Laboratory test results: The serum estradiol and progesterone levels of the subjects in the experimental group were significantly increased (P < 0.05), indicating that the complex could regulate the endocrine system and activate ovarian function.

[0188] There were no significant changes in the sex hormone levels of the subjects in the control group.

[0189] Conclusion: The cell viability regeneration complex of the present invention showed good safety and effectiveness in human clinical trials. Through the synergistic effect of various bioactive components, it could significantly improve problems such as relaxation, dryness, and atrophy of the female reproductive system, regulate the endocrine level, and enhance the overall health of the reproductive system. The experimental results showed that the complex was significantly superior to the placebo in humans and had broad application prospects.

[0190] In summary, the cell viability regeneration complex of the present invention, through the synergistic effect of various bioactive components, could activate the cell regeneration ability, promote the synthesis of elastin and collagen, regulate the endocrine system, and improve the overall health of the reproductive system. The experimental results showed that the complex showed good repair and regeneration effects at the cellular level, in animal models, and in human clinical trials, and had significant innovation and application value.

Claims

1. A cell vitality regeneration complex, characterized in that: The invention is composed of the following components in percentage by mass: 0.1-0.8% resveratrol, 0.1-1% cycloastragenol, 0.1-0.5% recombinant humanized type I collagen, 0.1-0.5% recombinant humanized type III collagen, 0.1-1% L-arginine, 0.1-1% citrulline, 0.1-1% lysine, 0.1-0.5% ginsenoside, 0.1-1% sodium hyaluronate, and the rest is purified water.

2. A cell vitality regeneration complex according to claim 1, characterized in that: It is composed of the following components in percentage by mass: 0.4% resveratrol, 0.5% cycloastragenol, 0.3% recombinant humanized type I collagen, 0.3% recombinant humanized type III collagen, 0.5% L-arginine, 0.5% citrulline, 0.5% lysine, 0.3% ginsenoside, 0.5% sodium hyaluronate, and the rest is purified water.

3. A method for preparing a cell vitality regeneration complex, characterized in that: For preparing a cell vitality regeneration complex as claimed in claim 1, the method is specifically implemented according to the following steps: S1. Raw material preparation: Weigh each component according to the above mass percentage to ensure that the purity and quality of each component meet the requirements; S2. Dissolution: Dissolve resveratrol, cycloastragenol, ginsenosides, and sodium hyaluronate in purified water respectively and stir until completely dissolved; S3. Mixing: Mix the dissolved resveratrol, cycloastragenol, ginsenosides and sodium hyaluronate solution and stir evenly; S4. Add collagen: Add recombinant humanized type I collagen and recombinant humanized type III collagen to the mixture and stir until completely dissolved; S5. Add amino acids: Add L-arginine, citrulline and lysine to the mixture and stir until completely dissolved; S6. Homogenization: Homogenize the mixture to ensure uniform distribution of components; S7. Filtering: The homogenized mixed solution is filtered through a microporous filter membrane to remove impurities and obtain a clarified complex solution; S8. Filling: Fill the filtered complex solution into a sterile container and seal it for storage; S9. Sterilization: The filled compound is sterilized at low temperature to finally obtain a cell vitality regeneration compound.

4. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In the step S1, the plant-derived components are dried at 50-70° C. for 6-10 hours and crushed to a particle size of less than 40 mesh.

5. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: The dissolving temperature of S2 is 25-40°C, and a magnetic stirrer or mechanical stirring is used with a stirring speed of 200-400 rpm.

6. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In S3, the mixing temperature is 25-40°C; a paddle stirrer is used, and the stirring speed is 300-500 rpm.

7. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In the S4, the dissolution temperature is 25-40°C; the stirring speed is 100-200 rpm, and the dissolution time is 1-2 hours.

8. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In the S5, the dissolving temperature is 25-40° C., the stirring speed is 200-300 rpm, and the dissolving time is 10-20 minutes.

9. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In the S6, the homogenization pressure is 50-100 MPa, and the high-pressure homogenizer is used for 3-5 cycles; the treatment temperature is 25-40° C., which is controlled by jacket cooling.

10. The method for preparing a cell vitality regeneration complex according to claim 3, characterized in that: In the S7, the filtration is carried out through a 0.22-0.25 μm microporous filter membrane at a filtration temperature of 25-40° C. and a filtration pressure of 0.1-0.3 MPa, using nitrogen pressure filtration.

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