A biphasic antibacterial and antimicrobial gel for regulating vaginal microecological balance and its preparation method

Abstract

This invention belongs to the technical field of medical gels, specifically relating to a biphasic antibacterial and probiotic gel for regulating vaginal microecological balance and its preparation method. The gel is divided into phase A and phase B at a mass ratio of 1.5–2.5:1. Phase A contains antibacterial components such as chitosan, tea polyphenols, and lactoferrin, while phase B contains probiotic factors such as short-chain fructooligosaccharides, vitamin B complex, and sodium hyaluronate. Phase A has a pH of 3.8–4.4, and phase B has a pH of 4.5–5.3, forming stable semi-solid gels that are individually packaged for use. Through the synergistic ratio of the three antibacterial components and the biphasic structure separating them from the beneficial components, a "first inhibit, then nourish" ecological intervention pathway is formed, effectively inhibiting pathogens such as Gardnerella vaginalis and Escherichia coli, promoting the colonization and reconstruction of lactobacilli, and achieving precise regulation and functional restoration of the vaginal microecology.

Description

Technical Field

[0001] This invention belongs to the technical field of medical gels, specifically relating to an antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance and its preparation method. Background Technology

[0002] The vaginal microecological system is a crucial barrier for women's reproductive health, composed of diverse microbial communities. A dominant flora, primarily lactobacilli, maintains the acidic environment of the vagina, thereby inhibiting the invasion and colonization of pathogenic microorganisms. In recent years, with changes in lifestyle, antibiotic overuse, and environmental factors, vaginal microecological imbalance has become increasingly prominent, easily leading to various gynecological infections such as bacterial vaginosis and yeast infections, seriously affecting women's quality of life and reproductive health. Therefore, regulating the vaginal microecology, promoting the growth of beneficial bacteria, and inhibiting the proliferation of harmful bacteria have become important directions in current gynecological treatment and care.

[0003] Currently, clinical methods for regulating vaginal microecology mainly include simple antibacterial therapy, exogenous probiotic supplementation, and the use of pH-regulating products. While antibiotics can rapidly eliminate pathogens, they can cause broad-spectrum bacterial death, exacerbating microbial imbalance and posing a risk of drug resistance. Exogenous probiotic supplementation suffers from low strain survival rates and poor colonization ability. Some commercially available pH-regulating products only provide short-term relief and cannot fundamentally restore the stable structure of the vaginal microecology. In addition, some traditional Chinese medicine suppositories or gels have been attempted in this area, but they generally suffer from a single mechanism of action, poor stability, and low patient compliance, failing to achieve dynamic and precise regulation of the vaginal microecology. Therefore, there is an urgent need to develop a novel formulation with both antibacterial and probiotic regulatory functions to more effectively rebuild and maintain the microecology.

[0004] To address these challenges, developing a functional formulation that can simultaneously inhibit harmful bacteria and promote the growth of beneficial bacteria is of significant practical importance and promising application prospects. Such a formulation should not only possess comprehensive regulatory capabilities in restoring microecological balance but also exhibit good biocompatibility and ease of use to meet the dual needs of clinical practice and routine health management. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a biphasic gel for regulating vaginal microecological balance, comprising two separate phases, A and B. Phase A is the antibacterial phase, and phase B is the probiotic phase. The two phases are used in combination at a mass ratio of A phase:B phase of (1.5–2.5):1.

[0006] Phase A comprises, by mass percentage: chitosan 0.4%–1.0%; tea polyphenols 0.1%–0.25%; lactoferrin 0.15%–0.4%; disodium EDTA 0.05%–0.2%; glycerol 0.2%–0.5%; carbomer 0.4%–1.5%; the balance being deionized water.

[0007] Phase B, by mass percentage, comprises: 1.5%–3.5% short-chain fructooligosaccharides; 0.03%–0.1% vitamin B complex; 0.015%–0.05% zinc gluconate; 0.3%–0.8% sodium hyaluronate; 0.8%–2.0% polyvinyl alcohol; 0.2%–0.8% propylene glycol; the balance being deionized water.

[0008] in:

[0009] In phase A, the mass ratio of chitosan, tea polyphenols and lactoferrin is (5-7):(1-2):(2-3);

[0010] In phase B, the mass ratio of short-chain fructooligosaccharides, vitamin B complex, and zinc gluconate is (30-50):(1-2):(1-2).

[0011] The pH value of phase A is 3.8–4.4, and the pH value of phase B is 4.5–5.3;

[0012] Both phase A and phase B are semi-solid gel-like dispersion systems, and are placed in separate sealed packages.

[0013] As a preferred technical solution, the chitosan contained in phase A is an acid-soluble chitosan with a degree of deacetylation of 85% to 95% and a number-average molecular weight of 8 × 10⁻⁶. 4 ~1.2×10 5 Dalton exists in a molecularly dissolved state in an aqueous phase with a pH value below 4.5 during the preparation of phase A, and does not form visible precipitates or particulate phases in the gel system.

[0014] As a preferred technical solution, the mass ratio of carbomer to glycerol contained in phase A is (1-3):1, and the carbomer forms a continuous three-dimensional cross-linked structure after neutralization treatment, while the glycerol is embedded in the cross-linked structure in a uniformly dispersed state. Phase A exhibits non-Newtonian fluid characteristics at 25°C.

[0015] As a preferred technical solution, the mass ratio of chitosan, tea polyphenols and lactoferrin in phase A is further limited to 6:(1-1.5):(2-2.5), and the three are added simultaneously and uniformly dispersed in the gel network formed by carbomer during the preparation of phase A.

[0016] As a preferred technical solution, the short-chain fructooligosaccharides contained in the B phase have an average degree of polymerization of 2 to 4 and a purity of not less than 95%. They exist in a completely dissolved state during the preparation of the B phase and are uniformly distributed in the continuous phase formed by polyvinyl alcohol.

[0017] As a preferred technical solution, regarding the polyvinyl alcohol and sodium hyaluronate contained in phase B, the polyvinyl alcohol has a degree of alcoholysis of 85%–89% and a number-average molecular weight of 2 × 10⁻⁶. 4 ~4×10 4 The number average molecular weight of Dalton's partially hydrolyzed polyvinyl alcohol and sodium hyaluronate is 5 × 10⁻⁶. 4 ~2×10 5 Dalton and Dalton form a cross-linked viscoelastic network structure in phase B at a mass ratio of 1.5:1 to 3:1, which improves the overall consistency and bioadhesion properties.

[0018] As a preferred technical solution, the vitamin B complex in phase B is composed of thiamine, riboflavin, pyridoxine and nicotinamide, with the mass ratio of the four in phase B being (2-4):(1-2):(2-3):(3-6), and is added under the condition that the temperature does not exceed 40°C during the preparation of phase B.

[0019] This invention also provides a method for preparing the aforementioned antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance, comprising the following steps:

[0020] S1 and A phase matrix pretreatment steps:

[0021] Slowly add carbomer to deionized water and stir at 300-500 rpm for 30-60 minutes at 20-30°C to fully disperse and complete the initial swelling of carbomer. Then add disodium EDTA and continue stirring for 10-20 minutes to form a homogeneous A-phase matrix solution.

[0022] S2, A-phase active component introduction steps:

[0023] In the A-phase matrix solution, chitosan, lactoferrin and tea polyphenols are added sequentially at 45℃~60℃, with an interval of no less than 5 minutes between each addition. After each addition, stirring is maintained for 10~20 minutes to ensure that the chitosan, lactoferrin and tea polyphenols are uniformly dispersed in the A-phase matrix. Then glycerol is added and stirring is continued for 15~30 minutes.

[0024] S3, Phase A pH adjustment and defoaming steps:

[0025] Add a buffer solution to the A-phase system after the components have been introduced to adjust the pH value to the range of 3.8 to 4.4, and then perform vacuum degassing for 10 to 20 minutes under the condition of -0.08 to -0.10 MPa to obtain the A-phase gel;

[0026] S4 and B phase matrix dissolution steps:

[0027] Polyvinyl alcohol is added to deionized water, heated to 80℃~90℃ and stirred for 30~60 minutes while maintaining this temperature until the polyvinyl alcohol is completely dissolved. Then it is naturally cooled to below 40℃ to form a B-phase matrix solution.

[0028] S5 and B phase active component compounding steps:

[0029] In the B-phase matrix solution, short-chain fructooligosaccharides, zinc gluconate, vitamin B complex, sodium hyaluronate and propylene glycol were added sequentially at 30℃~40℃. After each component was added, the mixture was stirred for 10~20 minutes to obtain a uniformly dispersed B-phase mixed system.

[0030] S6, Phase B pH adjustment and defoaming steps:

[0031] The pH of the B-phase mixture was adjusted to be within the range of 4.5 to 5.3, and then vacuum degassing was performed for 10 to 20 minutes under a pressure of -0.08 to -0.10 MPa to obtain the B-phase gel.

[0032] S7. Cavity filling steps:

[0033] The A-phase gel and B-phase gel are respectively filled into physically isolated dual-cavity packaging containers and sealed to obtain the antibacterial and antimicrobial biphase gel product.

[0034] As a preferred technical solution, in step S2, chitosan is added by pre-dissolving chitosan in an aqueous phase with a pH of 3.5 to 4.0 before introducing it into the A phase matrix solution, and the addition rate of the chitosan solution is controlled within the range of 0.5 to 2.0 mL / min.

[0035] As a preferred technical solution, after the polyvinyl alcohol is dissolved in step S4, it needs to be kept under stirring and naturally cooled to 35℃~40℃. In step S5, the sodium hyaluronate is added to the B phase mixture after the short-chain fructooligosaccharide and vitamin B complex, and the stirring time after the sodium hyaluronate is added is not less than 20 minutes.

[0036] Beneficial effects:

[0037] This invention, through a dual-cavity physical isolation design of phases A and B, achieves for the first time the structural separation and synergistic regulation of antibacterial and probacterial components, avoiding the technical problems of antagonistic interactions and reduced stability of active ingredients in traditional single-phase gels. This structure not only ensures the physicochemical stability of the two phases but also, through a sequential release mechanism in practical applications, first eliminates pathogenic bacteria and then supports the recovery and proliferation of dominant bacteria, realizing a complete regulatory chain from disruption to recovery to consolidation, demonstrating excellent ecological closed-loop construction capabilities.

[0038] Furthermore, the synergistic ratio of chitosan, tea polyphenols, and lactoferrin is introduced into phase A, supplemented by a three-dimensional gel network structure constructed with carbomer. This allows the active ingredients to be released uniformly in a stable state under slightly acidic conditions, effectively enhancing the adhesion and inhibition of opportunistic pathogens such as Gardnerella vaginalis and Escherichia coli, and strengthening the mucosal barrier function of local tissues. The precise control of this synergistic ratio avoids common problems such as irritation or insufficient activity of single components, demonstrating significant targeting and selectivity.

[0039] Furthermore, phase B uses short-chain fructooligosaccharides as the core nutrient substrate, combined with B vitamins and sodium hyaluronate to form a complex viscoelastic structure, which significantly improves the adhesion and colonization ability and metabolic activity of probiotics such as lactobacillus, thereby enhancing their acid production capacity and self-sustaining mechanism; in particular, the precise control of phase B pH and the presence of high-purity fructooligosaccharides in a soluble state improve the efficiency and duration of probiotic community recovery, avoiding technical problems such as unstable components and dilution by the host environment in traditional probiotic factor formulations. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the preparation method of the present invention;

[0041] Figure 2 This is a schematic diagram illustrating the antibacterial effect of the present invention;

[0042] Figure 3 This is a schematic diagram illustrating the antibacterial effect of the present invention;

[0043] Figure 4 This is a schematic diagram illustrating the pH stability effect of the present invention;

[0044] Figure 5 This is a schematic diagram illustrating the viscosity change rate effect of the present invention. Detailed Implementation

[0045] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0046] Experimental group 1

[0047] This experimental group provides a biphasic antibacterial and antimicrobial gel for regulating vaginal microecological balance. Its formulation design and preparation process are based on... Figure 1 As shown, the details are as follows:

[0048] I. Preparation of Phase A antibacterial gel:

[0049] (1) The formulation of phase A (by mass percentage) is as follows:

[0050] Chitosan: 0.4%; Tea polyphenols: 0.1%; Lactoferrin: 0.15%; Disodium EDTA: 0.05%; Glycerin: 0.2%; Carbomer: 0.4%; Balance: Deionized water.

[0051] (2) The specific preparation steps of phase A are as follows:

[0052] S1. Take 90 mL of deionized water and place it in a clean stirrer. Slowly add 0.4 g of carbomer at 25°C and stir at 400 rpm for 40 minutes. After the carbomer is fully dispersed and initially swollen, add 0.05 g of disodium EDTA and continue stirring for 15 minutes to form a homogeneous phase A matrix solution.

[0053] S2. Dissolve 0.4 g of chitosan in 10 mL of a buffer aqueous phase with a pH of 3.8, maintaining the solution temperature at 50°C. Slowly add the solution to phase A matrix at a rate of 1 mL / min and stir for 10 minutes. Then add 0.15 g of lactoferrin and 0.1 g of tea polyphenols sequentially, stirring for 15 minutes after each addition to ensure thorough dispersion. Finally, add 0.2 g of glycerol and continue stirring for 20 minutes.

[0054] S3. Adjust the pH to 4.0 using citrate-sodium citrate buffer, and perform vacuum degassing at -0.09 MPa for 15 minutes to obtain a transparent and homogeneous phase A gel with a stable pH of 4.0.

[0055] II. Preparation of Phase B Promoting Gel:

[0056] (1) The formulation of phase B (mass percentage) is as follows:

[0057] Short-chain fructooligosaccharides: 1.5%; Vitamin B complex: 0.03% (including thiamine: 0.012%, riboflavin: 0.006%, pyridoxine: 0.006%, nicotinamide: 0.006%); Zinc gluconate: 0.015%; Sodium hyaluronate: 0.3%; Polyvinyl alcohol: 0.8%; Propylene glycol: 0.2%; Balance: Deionized water.

[0058] (2) The specific preparation steps of phase B are as follows:

[0059] S4. Add 0.8 g of polyvinyl alcohol to 90 mL of deionized water, heat to 85°C, stir for 40 minutes until completely dissolved, and then allow to cool naturally to 38°C to form a transparent B-phase matrix solution.

[0060] S5. Add 1.5 g of short-chain fructooligosaccharides, 0.015 g of zinc gluconate, 0.03 g of vitamin B complex, 0.3 g of sodium hyaluronate, and 0.2 g of propylene glycol in sequence. Stir for 15 minutes after each addition to ensure uniform dispersion. Add sodium hyaluronate after the vitamin B complex, and continue stirring for 20 minutes after adding sodium hyaluronate.

[0061] S6. Adjust the pH to 4.6 using lactate-sodium lactate buffer, and degas under vacuum for 15 minutes to obtain a homogeneous phase B gel with a stable pH of 4.6.

[0062] III. Filling Steps:

[0063] S7. Inject the A-phase and B-phase gels into a dual-cavity plastic injector with a physical isolation structure, and after encapsulation, obtain the antibacterial and antimicrobial biphase gel product.

[0064] Experimental group 2

[0065] This experimental group provides a biphasic antibacterial and antimicrobial gel for regulating vaginal microecological balance. Its formulation and preparation process utilize the upper limits of each component, as detailed below:

[0066] I. Preparation of Phase A antibacterial gel:

[0067] (1) The formulation of phase A (by mass percentage) is as follows:

[0068] Chitosan: 1.0%; Tea polyphenols: 0.25%; Lactoferrin: 0.4%; Disodium EDTA: 0.2%; Glycerin: 0.5%; Carbomer: 1.5%; Deionized water: Balance (to bring to 100%).

[0069] (2) A phase preparation steps:

[0070] S1. Take 82.15 g of deionized water into a clean container, slowly add 1.5 g of carbomer at 30°C, stir at 500 rpm for 60 minutes to promote full swelling, then add 0.2 g of disodium EDTA and continue stirring for 20 minutes to form a high-viscosity A-phase matrix solution.

[0071] S2. Dissolve 1.0 g of chitosan in a pre-adjusted acidic aqueous phase at pH 4.4 (maintaining the temperature at 55℃), and slowly add it to the matrix at a rate of 1 mL / min, stirring for 10 minutes. Then add 0.4 g of lactoferrin and 0.25 g of tea polyphenols sequentially, stirring for 20 minutes after each addition; finally, add 0.5 g of glycerol and stir for 30 minutes.

[0072] S3. Adjust the pH of the system to 4.4, and then perform vacuum degassing at -0.10 MPa for 20 minutes to obtain a uniform and transparent A-phase gel with a pH of 4.4, which has good adhesion and rheological properties.

[0073] II. Preparation of Phase B Promoting Gel:

[0074] (1) The formulation of phase B (mass percentage) is as follows:

[0075] Short-chain fructooligosaccharides: 3.5%; Vitamin B complex: 0.1% (thiamine: 0.04%, riboflavin: 0.02%, pyridoxine: 0.03%, nicotinamide: 0.01%); Zinc gluconate: 0.05%; Sodium hyaluronate: 0.8%; Polyvinyl alcohol: 2.0%; Propylene glycol: 0.8%; Deionized water: balance.

[0076] (2) Preparation steps of phase B:

[0077] S4. Add 2.0 g of polyvinyl alcohol to approximately 91.65 g of deionized water, heat to 90°C and maintain this temperature while stirring for 60 minutes until completely dissolved; allow to cool naturally to 38°C to form a high-concentration B-phase matrix solution.

[0078] S5. At 38°C, add 3.5 g of short-chain fructooligosaccharides, 0.05 g of zinc gluconate, 0.1 g of vitamin B complex, 0.8 g of sodium hyaluronate, and 0.8 g of propylene glycol sequentially. After each component is added, stir for 20 minutes to ensure complete dissolution and uniform distribution.

[0079] S6. Adjust the pH to 5.3, and then perform vacuum degassing at -0.10 MPa for 20 minutes to obtain a highly active B-phase gel with a stable pH of 5.3, exhibiting good stability and distribution of probiotic substrates.

[0080] III. Filling Steps:

[0081] S7. Inject the A-phase and B-phase gels into a dual-cavity plastic injector with a physical isolation structure, and after encapsulation, obtain the antibacterial and antimicrobial biphase gel product.

[0082] Experimental group 3

[0083] This experimental group provides a biphasic gel for regulating vaginal microecological balance, which inhibits and promotes bacterial growth. The distribution ratio and process parameters are designed using the optimal values ​​in the middle of the parameter range. The preparation process is as follows:

[0084] I. Preparation of Phase A antibacterial gel:

[0085] (1) The formulation of phase A (by mass percentage) is as follows:

[0086] Chitosan: 0.7%; Tea polyphenols: 0.18%; Lactoferrin: 0.3%; Disodium EDTA: 0.1%; Glycerin: 0.35%; Carbomer: 1.0%; Deionized water: Balance (to bring to 100%).

[0087] (2) The preparation process of phase A is as follows:

[0088] S1. Slowly add 1.0 g of carbomer to 89.37 g of deionized water and stir at 400 rpm for 45 minutes at 25°C. After the carbomer has completely swollen, add 0.1 g of disodium EDTA and continue stirring for 15 minutes to form a homogeneous phase A matrix solution.

[0089] S2. Dissolve 0.7 g of chitosan in 10 mL of a buffer solution with a pH of 4.0. Heat to 50°C and maintain molecular-level dissolution. Add the solution to phase A at a rate of 1 mL / min and stir for 10 minutes. Then add 0.3 g of lactoferrin and 0.18 g of tea polyphenols sequentially, stirring for 15 minutes after each addition to ensure thorough dispersion. Finally, add 0.35 g of glycerol and stir for 25 minutes.

[0090] S3. Adjust the pH to 4.1 using citric acid / sodium citrate buffer, and perform vacuum degassing at -0.09 MPa for 15 minutes to obtain a stable and transparent A-phase gel, which is pale yellow and translucent, with good rheological properties and stable pH.

[0091] II. Preparation of Phase B Promoting Gel:

[0092] (1) The formulation of phase B (mass percentage) is as follows:

[0093] Short-chain fructooligosaccharides: 2.5%; Vitamin B complex: 0.07% (thiamine: 0.025%, riboflavin: 0.015%, pyridoxine: 0.015%, nicotinamide: 0.015%); Zinc gluconate: 0.03%; Sodium hyaluronate: 0.55%; Polyvinyl alcohol: 1.4%; Propylene glycol: 0.5%; Deionized water: balance.

[0094] (2) The preparation process of phase B is as follows:

[0095] S4. Add 1.4 g of polyvinyl alcohol to approximately 94.95 g of deionized water, heat to 85°C, stir for 45 minutes until completely dissolved, and cool to below 40°C to form phase B matrix solution.

[0096] S5. At 35°C, add 2.5 g of short-chain fructooligosaccharides, 0.03 g of zinc gluconate, 0.07 g of vitamin B complex, 0.55 g of sodium hyaluronate, and 0.5 g of propylene glycol sequentially. Stir each component for 15–20 minutes to ensure complete dissolution and dispersion. The order of addition of vitamin B complex and sodium hyaluronate has been optimized to ensure viscoelastic structure and activity stability.

[0097] S6. Adjust the pH to 4.9 using lactate / sodium lactate buffer, and then perform vacuum degassing at -0.09 MPa for 15 minutes to obtain a uniform, clear B-phase gel with moderate viscosity, suitable for probiotic colonization.

[0098] III. Filling Steps:

[0099] S7. Inject the A-phase and B-phase gels into a dual-cavity plastic injector with a physical isolation structure, and after encapsulation, obtain the antibacterial and antimicrobial biphase gel product.

[0100] Control group 1 (lacking synergistic components)

[0101] To verify the importance of the synergistic ratio of the antibacterial components in phase A of this invention, a control group 1 was set up. In phase A, the lactoferrin component was omitted, and only chitosan and tea polyphenols were retained. The remaining components and steps were the same as those in experimental group 3, and were used to observe changes in antibacterial stability and tissue affinity.

[0102] I. The formulation of phase A (by mass percentage) is as follows:

[0103] Chitosan: 0.7%; Tea polyphenols: 0.18%; Lactoferrin: None; Disodium EDTA: 0.1%; Glycerin: 0.35%; Carbomer: 1.0%; Deionized water: Balance (to bring to 100%).

[0104] A-phase preparation steps:

[0105] S1. Weigh 89.67 g of deionized water, add 1.0 g of carbomer, stir at 25℃ and 400 rpm for 45 minutes, add 0.1 g of disodium EDTA, and continue stirring for 15 minutes to form phase A matrix.

[0106] S2. Dissolve 0.7 g chitosan in 10 mL of buffer water with pH 4.0, heat to 50°C and slowly add the matrix, stirring for 10 minutes; then add 0.18 g tea polyphenols and stir for 15 minutes; add 0.35 g glycerol and continue stirring for 20 minutes.

[0107] S3. Adjust the pH to 4.1, degas under vacuum at -0.09 MPa for 15 minutes to obtain a pale yellow phase A gel.

[0108] II. Phase B is the same as experimental group 3, without any modifications.

[0109] 3. The product filling method is the same as that of Experiment Group 3, using a dual-chamber syringe for independent packaging.

[0110] Control group 2 (synergistic ratio exceeds the limits defined in this invention)

[0111] To verify the rationality and effectiveness of the mass ratio of chitosan, tea polyphenols, and lactoferrin specified in this invention (preferably within the range of 6:(1-1.5):(2-2.5)), a control group 2 was set up. In phase A, the above three synergistic antibacterial components were retained, but a ratio deviating from the scope of this invention was used: the mass ratio of chitosan, tea polyphenols, and lactoferrin was 3:1:5, meaning the chitosan ratio was too low and the lactoferrin ratio was too high. Other components and preparation steps remained consistent with experimental group 3.

[0112] I. The formulation of phase A (by mass percentage) is as follows:

[0113] Chitosan: 0.3%; Tea polyphenols: 0.1%; Lactoferrin: 0.5%; Disodium EDTA: 0.1%; Glycerin: 0.35%; Carbomer: 1.0%; Deionized water: balance.

[0114] A-phase preparation steps:

[0115] S1. Weigh 89.65 g of deionized water, slowly add 1.0 g of carbomer, stir at 25°C for 45 minutes, then add 0.1 g of disodium EDTA and continue stirring for 15 minutes.

[0116] S2. Dissolve 0.3 g chitosan in a pH 4.0 buffer solution, heat to 50°C and add to the system, stirring for 10 minutes; then add 0.5 g lactoferrin and 0.1 g tea polyphenols in sequence, stirring for 15-20 minutes each; finally add 0.35 g glycerol and stir for 20 minutes.

[0117] S3. Adjust the pH to 4.1, and after vacuum degassing for 15 minutes, phase A gel is obtained. The color is slightly milky white and the viscosity is significantly increased.

[0118] Phase B is exactly the same as in Experimental Group 3, and no modifications are made. The dual-cavity packaging steps are also the same.

[0119] Control group 3 (lacking synergistic components of tea polyphenols)

[0120] To verify the necessity of tea polyphenols in the synergistic system of chitosan, tea polyphenols and lactoferrin in phase A of this invention, a control group 3 was set up. In phase A, the tea polyphenol component was removed, while chitosan and lactoferrin were retained. The remaining components, parameters and preparation steps were kept the same as those in experimental group 3.

[0121] I. The formulation of phase A (by mass percentage) is as follows:

[0122] Chitosan: 0.7%; Tea polyphenols: None; Lactoferrin: 0.3%; Disodium EDTA: 0.1%; Glycerin: 0.35%; Carbomer: 1.0%; Deionized water: Balance (to bring to 100%).

[0123] A-phase preparation steps:

[0124] S1. Weigh 89.55 g of deionized water and slowly add 1.0 g of carbomer at 25°C. Stir at 400 rpm for 45 minutes to fully disperse and complete the initial swelling. Then add 0.1 g of disodium EDTA and continue stirring for 15 minutes to form a homogeneous phase A matrix solution.

[0125] S2. Dissolve 0.7 g of chitosan in 10 mL of a buffer aqueous phase with a pH of 4.0. Heat the solution to 50 °C and maintain the molecular-level dissolution state. Then slowly add the solution to phase A matrix at a rate of 1 mL / min and stir for 10 minutes. Then add 0.3 g of lactoferrin and continue stirring for 15 minutes. Finally, add 0.35 g of glycerol and stir for 25 minutes to make the system homogeneous.

[0126] S3. The pH of phase A was adjusted to 4.1 using a citric acid / sodium citrate buffer system, and vacuum degassing was performed for 15 minutes under -0.09 MPa conditions to obtain phase A gel.

[0127] II. The composition and preparation steps of phase B are exactly the same as those of experimental group 3, without any adjustments.

[0128] 3. The filling method is the same as that of Experimental Group 3, and a dual-cavity physical isolation packaging structure is used for independent sealing.

[0129] Control group 4 (based on existing technology monophasic antibacterial gel solution)

[0130] To further compare the differences between this invention and existing technologies in terms of functional structure and microecological regulation capabilities, a control group (4) was set up, constructing a control product that did not distinguish between antibacterial and probacterial phases and whose components were mixed in a single-phase system. This scheme did not distinguish between phase A and phase B, nor did it form a synergistic structure.

[0131] I. The single-phase gel formulation (by weight percentage) is as follows:

[0132] Chitosan: 0.6%; Lactoferrin: 0.3%; Tea polyphenols: 0.2%; Zinc gluconate: 0.02%; Polyvinyl alcohol: 1.5%; Glycerin: 0.4%; Carbomer: 0.8%; Propylene glycol: 0.5%; Deionized water: balance.

[0133] II. Preparation process:

[0134] S1. Add 0.8 g of carbomer and 1.5 g of polyvinyl alcohol to 90 mL of deionized water in sequence, and stir at 80°C for 45 minutes until completely dissolved.

[0135] S2. After cooling to 40℃, add 0.6 g chitosan (pre-dissolved), 0.3 g lactoferrin, 0.2 g tea polyphenols, 0.02 g zinc gluconate, 0.4 g glycerol and 0.5 g propylene glycol in sequence, and mix the components for 15-20 minutes.

[0136] S3. Adjust the pH to 4.5, degas for 15 minutes to obtain a single-phase gel, and seal it in a standard tube package.

[0137] Comparative experiment:

[0138] To verify the synergistic effect and technical advantages of the antibacterial and probiotic biphasic gel described in this invention in regulating the vaginal microecological environment, the following in vitro model experiments were set up to systematically compare experimental groups 1-3 with control groups 1-4.

[0139] 1. Experimental objective:

[0140] Assess the differences in performance among the gels in the following aspects:

[0141] Sustained inhibition of opportunistic pathogens (Gardnerella vaginalis, Escherichia coli, Candida albicans);

[0142] It enhances the protection and restoration of dominant probiotics (Lactobacillus);

[0143] pH stabilization effect and fluctuation range in a simulated vaginal environment;

[0144] Physical stability (phase separation, precipitation, viscosity change) under temperature and humidity storage conditions.

[0145] 2. The strains and culture conditions are shown in Table 1:

[0146] Table 1 Experimental bacterial strains and culture conditions

[0147]

[0148] III. Experimental Grouping and Sample Preparation:

[0149] Phase A and Phase B gels were prepared according to the formulations of experimental groups 1-3 and control groups 1-4, respectively. The finished products were uniformly packaged in 0.5 mL double-lumen syringes and mixed immediately before use. Control group 4 was a traditional single-phase gel and did not require mixing. All samples were stored at 4°C and brought to room temperature before use.

[0150] IV. Test Methods:

[0151] 1. Antibacterial persistence test (liquid plate dilution method):

[0152] The target pathogens were cultured to the logarithmic growth phase and diluted to approximately 10⁻⁶. 6 CFU / mL; Take 0.2 mL of each sample and mix it in 1.8 mL of simulated vaginal secretion (containing mucin, salt ions, lactate buffer system, pH=4.5); Add the target bacterial culture, mix and culture in an anaerobic bottle / shaker, and incubate at 37℃; Take samples every 12 hours, perform serial dilutions and plate, count the number of surviving colonies, and observe for 72 h; Compare the growth and decline trends of bacterial communities and the persistence of inhibition under different samples.

[0153] 2. Probiotic protection and colonization ability test:

[0154] Extracellular matrix proteins (such as collagen IV) were coated onto 96-well plates; gel samples were added and co-incubated with lactobacilli to simulate the cervical mucosal surface environment; after 24 h, the ability of bacterial film formation was assessed by crystal violet staining or by scraping and counting colonized bacteria; and a co-culture control of mixed Gardnerella vaginalis was performed to assess whether there were synergistic or antagonistic effects.

[0155] 3. pH stability test:

[0156] Each group of gels (0.5 mL) was added to simulated vaginal fluid; the solution was placed in a 37℃ incubator, and the pH of the solution was measured at 0 h, 6 h, 12 h, 24 h, and 48 h; the initial pH adjustment ability and the time span of stability maintenance of different groups of samples were compared.

[0157] 4. Gel appearance stability test:

[0158] Each group of samples was stored at 25℃ (room temperature) and 40℃ (accelerated temperature) for 30 days respectively; physical changes such as gel layering, precipitation, bubble regeneration, and discoloration were observed; the trend of shear viscosity change was measured using a rotational rheometer to assess physical stability.

[0159] Experimental data:

[0160] 1. Results of the antibacterial persistence test (48h colony forming units CFU / mL, log value), as shown in Table 2:

[0161] Table 2 Results of Antibacterial Duration Test

[0162]

[0163] Note: The initial bacterial concentration was 6.0 log CFU / mL. The lower the value, the stronger the antibacterial effect.

[0164] 2. Probiotic (Lactobacillus) colonization rate (unit: %), as shown in Table 3:

[0165] Table 3. Results of Probiotic (Lactobacillus) Colonization Rate Test

[0166]

[0167] 3. pH stability (initial pH 4.5, unit: change in pH ΔpH), as shown in Table 4:

[0168] Table 4 pH stability test results

[0169]

[0170] 4. Storage stability (indicators observed after 30 days of accelerated aging at 40℃), as shown in Table 5:

[0171] Table 5 Storage stability test results

[0172]

[0173] Experimental data analysis:

[0174] Based on the experimental data from the experimental and control groups, a comprehensive analysis of each group's antibacterial ability, probiotic protection, pH regulation stability, and storage stability is conducted as follows:

[0175] 1. Regarding the duration of antibacterial activity:

[0176] Experimental groups 1-3 all effectively inhibited the growth of Gardnerella vaginalis, Escherichia coli, and Candida albicans within 48 hours, and the colony count decreased significantly compared to the initial value. Figure 2 As shown, the three components synergistically exhibit good antibacterial efficacy. In particular, experimental group 3, with its optimal ratio, maintained the survival rate of the three conditionally pathogenic bacteria below 2.5 log CFU / mL for 48 hours, demonstrating a strong and sustained antibacterial effect. In contrast, control groups 1 and 3, lacking lactoferrin or tea polyphenols respectively, only achieved initial antibacterial activity, with the number of pathogens rebounding rapidly within 48 hours. Control group 2, although containing all components, had an unbalanced ratio, resulting in a high concentration of lactoferrin covering the antibacterial spectrum, which weakened the chitosan effect, making its antibacterial effect inferior to the embodiment of this invention. Control group 4, although derived from existing technology, suffers from unstable antibacterial effects due to the easy interference between components in its single-phase structure.

[0177] 2. Regarding probiotic colonization ability:

[0178] In the simulated cervical adhesion model, experimental groups 1-3 showed significant protective and promoting effects against lactobacilli, such as... Figure 3 As shown, the colonization rate at 48 hours was higher than 65% in all groups, with experimental group 3 reaching as high as 89.4%, significantly better than the other groups. This advantage stems from the complex viscoelastic network structure constructed from B-phase fructooligosaccharides, B vitamins, and sodium hyaluronate, which provides nutritional support for probiotics and enhances their adhesion stability in the gel system. Control groups 1 and 3 had significantly lower colonization rates due to incomplete synergistic structures, leading to inhibition of probiotics by the antibacterial components or environmental incompatibility. In control group 4, the mixture of antibacterial and probiotic-promoting components caused significant interference, limiting the growth of probiotics.

[0179] 3. Regarding pH stability regulation capability:

[0180] like Figure 4 As shown, the experimental groups of this invention can effectively maintain the pH of the simulated environment stably within the range of 4.0 to 5.0, and the fluctuation range is less than 0.4 within 48 hours, with experimental group 3 showing the smallest fluctuation at only 0.24. This good pH regulation function can maintain the stability of the vaginal microecology and inhibit the growth of pH-sensitive pathogens. In contrast, control groups 2 and 3 exhibited uneven acid-base regulation, resulting in a wider pH fluctuation range and unstable intervention environment; while control group 4, although initially capable of regulation, struggled to maintain a stable plateau over time, potentially leading to bacterial replacement disorder.

[0181] 4. Regarding gel storage stability:

[0182] like Figure 5 As shown, in the 30-day accelerated aging experiment at 40℃, experimental groups 1-3 showed no obvious stratification or precipitation, and the viscosity change rate was controlled within 10%. In particular, the change in experimental group 3 was only +2.1%, indicating that the formulation system has good physical stability and is suitable for actual clinical applications. Control groups 1-3 showed varying degrees of protein aggregation, precipitation, and stratification, indicating poor storage stability; control group 4 also showed some viscosity fluctuation, indicating insufficient structural compatibility.

[0183] In conclusion, this invention significantly enhances the persistence of antibacterial activity and the recovery capacity of probiotics through the synergistic ratio control of antibacterial components and the physical separation design of a biphasic structure. It achieves an ecological intervention pathway of "inhibition followed by nourishment," demonstrating comprehensive performance far superior to existing technologies in regulating vaginal microecological balance. Experimental group 3, as the preferred scheme, showed the best performance in all core evaluation indicators, fully verifying the advanced nature and technical value of this invention in terms of functional synergy, structural rationality, and application effect.

[0184] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biphasic gel for regulating vaginal microecological balance, characterized in that, It includes two separate phases, A and B, where phase A is an antibacterial phase and phase B is a probiotic phase. The two phases are used in combination at a mass ratio of phase A:phase B of (1.5–2.5):

1. Phase A comprises, by mass percentage: chitosan 0.4%–1.0%; tea polyphenols 0.1%–0.25%; lactoferrin 0.15%–0.4%; disodium EDTA 0.05%–0.2%; glycerol 0.2%–0.5%; carbomer 0.4%–1.5%; the balance being deionized water. Phase B, by mass percentage, comprises: 1.5%–3.5% short-chain fructooligosaccharides; 0.03%–0.1% vitamin B complex; 0.015%–0.05% zinc gluconate; 0.3%–0.8% sodium hyaluronate; 0.8%–2.0% polyvinyl alcohol; 0.2%–0.8% propylene glycol; the balance being deionized water. in: In phase A, the mass ratio of chitosan, tea polyphenols and lactoferrin is (5-7):(1-2):(2-3); In phase B, the mass ratio of short-chain fructooligosaccharides, vitamin B complex, and zinc gluconate is (30-50):(1-2):(1-2). The pH value of phase A is 3.8–4.4, and the pH value of phase B is 4.5–5.3; Both phase A and phase B are semi-solid gel-like dispersion systems, and are placed in separate sealed packages.

2. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to claim 1, characterized in that, Regarding the chitosan contained in phase A, the chitosan is an acid-soluble chitosan with a degree of deacetylation of 85% to 95% and a number-average molecular weight of 8 × 10⁻⁶. 4 ~1.2×10 5 Dalton exists in a molecularly dissolved state in an aqueous phase with a pH value below 4.5 during the preparation of phase A, and does not form visible precipitates or particulate phases in the gel system.

3. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to claim 1, characterized in that, Regarding the carbomer and glycerol contained in the A phase, the mass ratio of carbomer to glycerol is (1~3):1, and the carbomer forms a continuous three-dimensional cross-linked structure after neutralization treatment, while the glycerol is embedded in the cross-linked structure in a uniformly dispersed state. The A phase exhibits non-Newtonian fluid characteristics at 25°C.

4. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to any one of claims 1 to 3, characterized in that, The mass ratio of chitosan, tea polyphenols and lactoferrin in phase A is further limited to 6:(1-1.5):(2-2.5), and the three are added simultaneously and uniformly dispersed in the gel network formed by carbomer during the preparation of phase A.

5. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to claim 1, characterized in that, The short-chain fructooligosaccharides contained in phase B have an average degree of polymerization of 2 to 4 and a purity of not less than 95%. They exist in a completely dissolved state during the preparation of phase B and are uniformly distributed in the continuous phase formed by polyvinyl alcohol.

6. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to claim 1 or 5, characterized in that, Regarding the polyvinyl alcohol and sodium hyaluronate contained in phase B, the polyvinyl alcohol has a degree of alcoholysis of 85%–89% and a number-average molecular weight of 2 × 10⁻⁶. 4 ~4×10 4 The number average molecular weight of Dalton's partially hydrolyzed polyvinyl alcohol and sodium hyaluronate is 5 × 10⁻⁶. 4 ~2×10 5 Dalton and Dalton form a cross-linked viscoelastic network structure in phase B at a mass ratio of 1.5:1 to 3:1, which improves the overall consistency and bioadhesion properties.

7. The antibacterial and antimicrobial biphasic gel for regulating vaginal microecological balance according to claim 1, characterized in that, The vitamin B complex in phase B consists of thiamine, riboflavin, pyridoxine and nicotinamide, with a mass ratio of (2-4):(1-2):(2-3):(3-6) in phase B, and is added under the condition that the temperature does not exceed 40°C during the preparation of phase B.

8. A method for preparing a biphasic antibacterial and antimicrobial gel for regulating vaginal microecological balance as described in any one of claims 1 to 7, characterized in that, Includes the following steps: S1, A phase matrix pretreatment steps: Slowly add carbomer to deionized water and stir at 300-500 rpm for 30-60 minutes at 20-30°C to fully disperse and complete the initial swelling of carbomer. Then add disodium EDTA and continue stirring for 10-20 minutes to form a homogeneous A-phase matrix solution. S2, A-phase active component introduction steps: In the A-phase matrix solution, chitosan, lactoferrin and tea polyphenols are added sequentially at 45℃~60℃, with an interval of no less than 5 minutes between each addition. After each addition, stirring is maintained for 10~20 minutes to ensure that the chitosan, lactoferrin and tea polyphenols are uniformly dispersed in the A-phase matrix. Then glycerol is added and stirring is continued for 15~30 minutes. S3, Phase A pH adjustment and defoaming steps: Add a buffer solution to the A-phase system after the components have been introduced to adjust the pH value to the range of 3.8 to 4.4, and then perform vacuum degassing for 10 to 20 minutes under the condition of -0.08 to -0.10 MPa to obtain the A-phase gel; S4 and B phase matrix dissolution steps: Polyvinyl alcohol is added to deionized water, heated to 80℃~90℃ and stirred for 30~60 minutes while maintaining this temperature until the polyvinyl alcohol is completely dissolved. Then it is naturally cooled to below 40℃ to form a B-phase matrix solution. S5 and B phase active component compounding steps: In the B-phase matrix solution, short-chain fructooligosaccharides, zinc gluconate, vitamin B complex, sodium hyaluronate and propylene glycol were added sequentially at 30℃~40℃. After each component was added, the mixture was stirred for 10~20 minutes to obtain a uniformly dispersed B-phase mixed system. S6, Phase B pH adjustment and defoaming steps: The pH of the B-phase mixture was adjusted to be within the range of 4.5 to 5.3, and then vacuum degassing was performed for 10 to 20 minutes under a pressure of -0.08 to -0.10 MPa to obtain the B-phase gel. S7. Cavity filling steps: The A-phase gel and B-phase gel are respectively filled into physically isolated dual-cavity packaging containers and sealed to obtain the antibacterial and antimicrobial biphase gel product.

9. The preparation method according to claim 8, characterized in that, In step S2, chitosan is added by pre-dissolving it in an aqueous phase with a pH of 3.5 to 4.0 before introducing it into the A phase matrix solution, and the addition rate of the chitosan solution is controlled within the range of 0.5 to 2.0 mL / min.

10. The preparation method according to claim 8, characterized in that, After the polyvinyl alcohol is dissolved in step S4, it needs to be kept under stirring and allowed to cool naturally to 35℃~40℃. In step S5, the sodium hyaluronate is added to the B phase mixture after the short-chain fructooligosaccharides and vitamin B complex, and the stirring time should be no less than 20 minutes after the sodium hyaluronate is added.

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