A product for improving vaginal flora balance and immunity and a method for preparing the same

Abstract

The application discloses a product for improving vaginal flora balance and improving immunity and a preparation method thereof, and belongs to the field of biological medicine. The product comprises an A-phase matrix and a B-phase active component, and realizes triple ecological regulation through three groups of synergistic components: directional plant cell compound liquid and specific private protection antibacterial peptide synergistically kill and protect bacteria, probiotics-PDRN super-assembled nanoparticles and transdermal repair peptides synergistically repair mucosa, and motherwort iridoid enriched liquid and PQQ-cyclodextrin inclusion compound synergistically enhance immunity. The product realizes long-acting slow release of active ingredients in the vaginal environment for 12-24 hours through the following innovations: a PDRN self-assembly super-assembled nanoparticle technology attached to the surface of plant lactobacillus 299v, and a 50-500nm layered microspherization controlled release technology with an encapsulation efficiency of greater than or equal to 85%. The product can quickly restore the vaginal microecological balance, significantly improve the local immunity, promote the mucosal repair, and reduce the risk of repeated infection.

Description

Technical Field

[0001] This invention relates to biomedicine, and more particularly to a product for improving vaginal flora balance and enhancing immunity, and its preparation method. Background Technology

[0002] According to data from the World Health Organization (WHO), approximately 75% of women worldwide will experience vaginal flora imbalance at least once in their lifetime, with bacterial vaginosis having a prevalence rate as high as 23%-29%. Vaginal microecological imbalance not only leads to common symptoms such as vulvar itching, abnormal vaginal discharge, odor, vaginitis, and cervical erosion, but is also closely related to serious consequences such as recurrent infections, pelvic inflammatory disease, and even infertility.

[0003] Existing feminine hygiene products have the following technical defects:

[0004] (1) Traditional herbal products mainly use single plant extracts such as Sophora flavescens and Cnidium monnieri, which only have basic antibacterial and cleansing functions, and cannot achieve systematic regulation of vaginal microecology, let alone improve the immunity of private parts.

[0005] (2) Physical barrier products form a physical isolation layer with alginate and the like. Although they have a certain effect on microecological restoration, they lack the triple synergistic mechanism of active sterilization, protection and inoculation.

[0006] (3) Ordinary prebiotic products only provide the nutritional substrate for probiotics and cannot directly supplement functional active ingredients, so they are slow to take effect and have limited effects.

[0007] (4) Existing products generally lack effective means for deep repair of the intimate mucosa and are insufficient in repairing pathological conditions such as mucosal damage and ulceration.

[0008] (5) The active ingredients have poor stability in the acidic environment of the vagina, low transdermal absorption efficiency, and short duration of action, making it difficult to achieve long-term care.

[0009] Therefore, there is an urgent need in this field for a feminine care product that can simultaneously achieve broad-spectrum sterilization, beneficial bacteria promotion, deep mucosal repair, and enhancement of intimate immunity, while also possessing a long-lasting sustained-release function. Summary of the Invention

[0010] Purpose of the Invention: The purpose of this invention is to provide a product and its preparation method for improving vaginal flora balance and enhancing immunity. This product has the following advantages: it combines broad-spectrum bactericidal action with the promotion of beneficial bacteria, rapidly restoring vaginal microecological balance; it deeply repairs the intimate mucosa, promoting the regeneration of damaged tissue; it enhances the self-immunity of the intimate area, reducing the risk of recurrent infections; it achieves long-lasting sustained release of active ingredients through intelligent controlled-release technology; and all ingredients are natural, safe, gentle, and non-irritating, suitable for daily intimate care.

[0011] Technical solution: A product for improving vaginal flora balance and enhancing immunity, said product comprising the following components by weight:

[0012] Phase A: 65-80 parts water, 0.5-2 parts carbomer U20, 3-8 parts glycerol, 0.2-1 parts matrine;

[0013] Phase B: 1-5 parts of directional plant cell compound solution, 1-5 parts of probiotic-PDRN super-assembled nanoparticles, 1-4 parts of Leonurus japonicus iridoid enrichment solution, 0.01-0.1 parts of PQQ-cyclodextrin inclusion complex, 1-5 parts of transdermal repair peptide, 1-5 parts of specific feminine hygiene antimicrobial peptide, and 1-5 parts of layered microsphere controlled-release carrier.

[0014] The product comprises three synergistic components:

[0015] Group 1: Targeted plant cell compound liquid and specific private care antimicrobial peptide, with a mass ratio of 1:0.5-1:2, synergistically achieve the dual effects of broad-spectrum bactericidal effect and beneficial bacteria promotion;

[0016] The second group consists of probiotic-PDRN super-assembled nanoparticles and transdermal repair peptides, with a mass ratio of 1:0.5-1:2, which synergistically achieve deep mucosal repair and cell regeneration.

[0017] The third group: Leonurus japonicus cyclohexene ether enrichment solution and PQQ-cyclodextrin inclusion complex, with a mass ratio of 1:0.005-1:0.05, synergistically achieves immune enhancement by improving microcirculation and activating mitochondria;

[0018] The method for preparing the layered microsphere controlled-release carrier is as follows: the active ingredient is dissolved in an organic solvent to form an oil phase; in the presence of a surfactant, the oil phase is dispersed in an aqueous phase to form a primary emulsion; the active ingredient is self-assembled into a nanoscale layered microsphere structure by evaporating the organic solvent; the microspheres have a particle size of 50-500 nm and an encapsulation rate of not less than 85%; and sustained release is achieved in a vaginal environment of pH 3.8-4.5 for 12-24 hours.

[0019] The specific feminine care antimicrobial peptide is MetaAMP12, an antimicrobial peptide with selective antibacterial activity, which has a killing effect on vaginal pathogens but no inhibition on lactobacilli.

[0020] The preparation method of the probiotic-PDRN superassembled nanoparticles is as follows: Lactobacillus plantarum 299v is mixed with PDRN solution with a concentration of 0.1-5 mg / mL at a mass ratio of 1:50, and incubated at pH 6.8 and 35℃ for 3 hours. PDRN molecules are self-assembled and attached to the surface of the bacteria by hydrogen bonding and electrostatic attraction to form superassembled units with a particle size of 80-150 nm. Each unit is loaded with no less than 500,000 PDRN molecules. The nanoparticles are then lyophilized and reconstituted before use.

[0021] The targeted plant cell compound solution is composed of extracts of Coptis chinensis, Phellodendron chinense, Sophora flavescens, Cnidium monnieri, Lonicera japonica, Forsythia suspensa, Leonurus japonicus, and Glycyrrhiza uralensis in a mass ratio of (1-2):(1-2):(2-3):(1-1.5):(1-2):(1-2):(1-1.5):(2-3), and its total flavonoid content is not less than 15wt%.

[0022] Furthermore, the specific feminine antimicrobial peptide has a minimum inhibitory concentration (MIC) of ≤8 μg / mL against Gardnerella vaginalis and a half-maximal inhibitory concentration (IC50) of ≥128 μg / mL against Lactobacillus.

[0023] Furthermore, the transdermal repair peptide is palmitoyl pentapeptide-4 or acetyl hexapeptide-8, which achieves targeted penetration into the deep folds of the vaginal mucosa by coupling with hyaluronic acid modified on the surface of the stacked microsphere controlled-release carrier.

[0024] Furthermore, the preparation process of the PQQ-cyclodextrin inclusion complex is as follows: PQQ and β-cyclodextrin are stirred in a water bath at 60°C for 2 hours at a molar ratio of 1:1.2, and then freeze-dried to obtain the inclusion complex. Its stability in a pH 4.0-5.0 environment is more than 3 times higher than that of free PQQ.

[0025] Furthermore, the final pH value of the product is adjusted to 3.8-4.5, the osmotic pressure is 260-320 mOsm / kg, which is isotonic with the physiological environment of the vagina, and it does not contain any synthetic preservatives.

[0026] This invention also provides a method for preparing the above-mentioned product for improving vaginal flora balance and enhancing immunity, comprising the following steps:

[0027] S1: Add the purified water required for phase A to the vacuum emulsifier, add carbomer U20, glycerol and matrine, heat to 85°C at a rate of 2°C / min, turn on the homogenizer and shear at 5000rpm for 15 minutes, and sterilize at a constant temperature for 10 minutes.

[0028] S2: When the temperature drops to 50℃, add the directional plant cell compound solution, the motherwort iridoid enrichment solution, and the PQQ-cyclodextrin inclusion complex in sequence, and stir at 200 rpm to dissolve.

[0029] S3: When the temperature drops to 42℃, slowly add the probiotic-PDRN super-assembled nanoparticles, transdermal repair peptides, specific feminine care antimicrobial peptides and layered microsphere controlled-release carrier suspension to the system, and control the stirring speed to ≤300rpm.

[0030] S4: Adjust the pH to 4.0 with lactic acid, test the viscosity and microbial limits, and after passing the test, filter through a 0.22μm microporous membrane for sterilization and then fill.

[0031] Furthermore, the method of adding the layered microsphere controlled-release carrier suspension in step S3 is as follows: a microfluidic injection pump is used to inject at a constant rate of 0.5 mL / min, and an ultrasonic probe (frequency 40 kHz) is set at the injection port for online dispersion to prevent microsphere aggregation.

[0032] Furthermore, the product is suitable for at least one of the following symptoms: vulvar itching, abnormal vaginal discharge, vaginal microecological imbalance, and cervicitis.

[0033] Beneficial effects:

[0034] (1) The targeted plant cell compound solution of the present invention is composed of extracts from eight plants, namely Coptis chinensis, Phellodendron chinense, Sophora flavescens, Cnidium monnieri, Lonicera japonica, Forsythia suspensa, Leonurus japonicus, and Glycyrrhiza uralensis, in a specific ratio. It has broad-spectrum antibacterial, anti-inflammatory and antipruritic effects, and estrogen-like effects that promote the growth of Lactobacillus. The specific antimicrobial peptide MetaAMP12 is a targeted antimicrobial peptide that precisely kills pathogenic bacteria while protecting beneficial bacteria. The two work synergistically at a mass ratio of 1:0.5-1:2 to achieve precise microecological regulation that "broadly kills bacteria without harming beneficial bacteria".

[0035] (2) This invention is the first to form a supramolecular structure by non-covalent self-assembly of PDRN and probiotics. Each probiotic carrier surface is assembled with no less than 500,000 PDRN molecules, and 1 mL of the formulation contains no less than 6.5 billion repair microspheres. This structure combines the flora regulation function of probiotics and the tissue repair function of PDRN, achieving synergistic treatment with "one sphere, two effects". Probiotic-PDRN superassembled nanoparticles assemble a large number of PDRN molecules on the surface of a single probiotic through intelligent self-assembly technology, forming a high-density repair unit to achieve DNA-level deep repair. Transdermal repair peptides promote the transdermal absorption of active ingredients and accelerate cell proliferation. The two work synergistically, with a mass ratio of 1:0.5-1:2, to achieve rapid healing and tissue regeneration of mucosal damage.

[0036] (3) This invention is the first to combine Leonurus japonicus iridoid enrichment with PQQ-cyclodextrin inclusion complex in the field of intimate care. Leonurus japonicus iridoid enrichment provides sufficient nutrition to mucosal cells by improving local microcirculation, while PQQ-cyclodextrin inclusion complex fundamentally improves cellular energy metabolism efficiency by activating mitochondrial biosynthesis. The two form a mitochondrial-targeted immune enhancement pathway of "nutritional supply + energy activation", which has breakthrough effects in intimate anti-aging, postpartum repair, menopausal discomfort and other scenarios. Leonurus japonicus iridoid enrichment is rich in active ingredients such as leonurine, stachydrine, and flavonoids, which improve blood circulation in the intimate area, nourish the mucosa, and have antibacterial and soothing effects. PQQ-cyclodextrin inclusion complex has excellent antioxidant capacity and unique mitochondrial regeneration function. The two work synergistically, with a mass ratio of 1:0.005-1:0.05, to significantly enhance intimate immunity through the dual pathway of improving microcirculation and activating cellular energy metabolism.

[0037] (4) This invention develops a nanoscale self-assembled stacked microsphere structure, encapsulating active ingredients in multilayered lipid / polymer alternating shells. The microspheres have a particle size of 50-500 nm and an encapsulation rate of ≥85%, achieving a stepwise sustained release over 12-24 hours in a vaginal pH environment of 3.8-4.5 through ionic strength response. This technology solves the industry problem of rapid loss of active ingredients in the vaginal environment and short duration of action.

[0038] (5) This invention is the first to propose and realize a triple ecological regulation strategy of "targeted plant cell compound liquid (bactericidal) + probiotic-PDRN super-assembled nanoparticles (bacterial seeding) + Leonurus japonicus iridoid enrichment liquid and PQQ-cyclodextrin inclusion complex synergistic (bacterial protection)". Unlike the traditional single antibacterial approach, this invention actively promotes the colonization of lactobacilli and maintains the weakly acidic environment of the vagina while killing pathogenic bacteria, thus rebuilding a healthy microecology from the root. Attached Figure Description

[0039] Figure 1 This is a schematic diagram illustrating the overall triple synergistic effect mechanism of the product of this invention.

[0040] Figure 2 This is a schematic diagram of the layered microsphere controlled-release structure and sustained-release process of the present invention.

[0041] Figure 3 This is a schematic diagram of the probiotic-PDRN superassembled nanoparticle structure of the present invention. Detailed Implementation

[0042] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0043] Example 1: Standard Formulation

[0044] Raw material formula (by weight):

[0045]

[0046] Preparation method:

[0047] Step S1: Add the required water (72.5 kg) for phase A to the vacuum emulsifier, add carbomer U20 (1.0 kg), glycerol (5.0 kg), and matrine (0.5 kg), heat to 85°C at a rate of 2°C / min, turn on the homogenizer and shear at 5000 rpm for 15 minutes, and sterilize at a constant temperature for 10 minutes.

[0048] Step S2: Turn off the heating and turn on the cooling water to cool down at a rate of 2℃ / min. When the temperature drops to 50℃, add the heat-stabilizing components in phase B in sequence: directional plant cell compound solution (3.0kg), Leonurus japonicus iridoid enrichment solution (2.0kg), and PQQ-cyclodextrin inclusion complex (0.05kg), and stir to dissolve at 200rpm.

[0049] Step S3: Continue cooling to 42℃, then slowly add probiotic-PDRN super-assembled nanoparticles (3.0kg), transdermal repair peptides (3.0kg), specific feminine care antimicrobial peptide MetaAMP12 (3.0kg), and layered microsphere controlled-release carrier suspension (3.0kg) to the system, controlling the stirring speed to ≤300rpm; the layered microsphere controlled-release carrier suspension is added by injecting it at a constant rate of 0.5 mL / min using a microfluidic injection pump, and setting an ultrasonic probe (frequency 40kHz) at the injection port for online dispersion to prevent microsphere aggregation;

[0050] Step S4: Adjust the pH to 4.1 with lactic acid, test the viscosity and microbial limits, and after passing the test, filter the solution through a 0.22μm microporous membrane for sterilization and then fill it into a container.

[0051] Example 2: High-efficacy concentrated form

[0052] Raw material formula (by weight):

[0053]

[0054] Preparation method:

[0055] Step S1: Add the required water (65.0 kg) for phase A to the vacuum emulsifying pot, add carbomer U20 (0.5 kg), glycerol (3.0 kg), and matrine (1.0 kg), heat to 85°C at a rate of 2°C / min, turn on the homogenizer and shear at 5000 rpm for 15 minutes, and sterilize at a constant temperature for 10 minutes.

[0056] Step S2: Turn off the heating and turn on the cooling water to cool down at a rate of 2℃ / min. When the temperature drops to 50℃, add the heat-stabilizing components in phase B in sequence: directional plant cell compound solution (5.0kg), Leonurus japonicus iridoid enrichment solution (4.0kg), and PQQ-cyclodextrin inclusion complex (0.1kg), and stir to dissolve at 200rpm.

[0057] Step S3: Continue cooling to 42℃, then slowly add probiotic-PDRN super-assembled nanoparticles (5.0kg), transdermal repair peptides (5.0kg), specific feminine care antimicrobial peptide MetaAMP12 (5.0kg), and layered microsphere controlled-release carrier suspension (5.0kg) to the system, controlling the stirring speed to ≤300rpm; the layered microsphere controlled-release carrier suspension is added by injecting it at a constant rate of 0.5 mL / min using a microfluidic injection pump, and setting an ultrasonic probe (frequency 40kHz) at the injection port for online dispersion to prevent microsphere aggregation;

[0058] Step S4: Adjust the pH to 3.85 with lactic acid, test the viscosity and microbial limits, and after passing the test, filter the product through a 0.22μm microporous membrane for sterilization and then fill it into a container.

[0059] Example 3: Routine Maintenance Type

[0060] Raw material formula (by weight):

[0061]

[0062] Preparation method:

[0063] Step S1: Add the required water (80.0 kg) for phase A to the vacuum emulsifying pot, add carbomer U20 (2.0 kg), glycerol (8.0 kg), and matrine (0.2 kg), heat to 85°C at a rate of 2°C / min, turn on the homogenizer and shear at 5000 rpm for 15 minutes, and sterilize at a constant temperature for 10 minutes;

[0064] Step S2: Turn off the heating and turn on the cooling water to cool down at a rate of 2℃ / min. When the temperature drops to 50℃, add the heat-stabilizing components in phase B in sequence: directional plant cell compound solution (1.0kg), Leonurus japonicus iridoid enrichment solution (1.0kg), and PQQ-cyclodextrin inclusion complex (0.01kg), and stir to dissolve at 200rpm.

[0065] Step S3: Continue cooling to 42℃, then slowly add probiotic-PDRN super-assembled nanoparticles (1.0kg), transdermal repair peptides (1.0kg), specific feminine care antimicrobial peptide MetaAMP12 (1.0kg), and layered microsphere controlled-release carrier suspension (1.0kg) to the system, controlling the stirring speed to ≤300rpm; the layered microsphere controlled-release carrier suspension is added by injecting it at a constant rate of 0.5 mL / min using a microfluidic injection pump, and setting an ultrasonic probe (frequency 40kHz) at the injection port for online dispersion to prevent microsphere aggregation;

[0066] Step S4: Adjust the pH to 4.4 with lactic acid, test the viscosity and microbial limits, and after passing the test, filter the solution through a 0.22μm microporous membrane for sterilization and then fill it into a container.

[0067] Comparative Example 1

[0068] The formulation is the same as in Example 1, except that it does not contain the specific feminine care antimicrobial peptide MetaAMP12, but is replaced with an equal amount of water.

[0069] Comparative Example 2

[0070] The formulation is the same as in Example 1, except that it does not contain probiotic-PDRN superassembled nanoparticles and is replaced with an equal amount of water.

[0071] Comparative Example 3

[0072] The formulation is the same as in Example 1, except that it does not contain PQQ-cyclodextrin inclusion complex and is replaced with an equal amount of water.

[0073] Comparative Example 4

[0074] The formulation is the same as that in Example 1, except that it does not contain the stacked microsphere controlled-release carrier and is replaced by an equal amount of water.

[0075] Comparative Example 5

[0076] The formulation is the same as in Example 1, except that the same amount of ordinary Lactobacillus plantarum 299v lyophilized powder is physically blended with free PDRN, and no self-assembled nanoparticles are prepared, instead of probiotic-PDRN super-assembled nanoparticles.

[0077] Comparative Example 6

[0078] The formulation is the same as in Example 1, except that an equal molar amount of free PQQ active pharmaceutical ingredient is used instead of the PQQ-cyclodextrin inclusion complex.

[0079] Effect verification test

[0080] 1. In vitro antibacterial test: The Oxford cup method was used to detect the diameter of the inhibition zone against Escherichia coli, Staphylococcus aureus, and Candida albicans in Examples 1-3 and Comparative Examples 1-6. The results are as follows:

[0081]

[0082] The results showed that the inhibition zones of Examples 1-3 exhibited a clear dose-dependent effect—the high-efficacy concentrated form (Example 2) showed the strongest inhibitory effect against Escherichia coli (30.5 mm), Staphylococcus aureus (28.2 mm), and Candida albicans (26.0 mm); the standard formulation (Example 1) was second; the daily maintenance form (Example 3) had the lowest concentration of antimicrobial peptides, resulting in a relatively small inhibition zone, but still maintained effective antimicrobial activity; the inhibition zone of Comparative Example 1 (lacking the specific feminine care antimicrobial peptide MetaAMP12) dropped sharply to 9.6-12.4 mm, a decrease of approximately 53-57% compared to Example 1, proving that MetaAMP12 is the absolute core of the broad-spectrum bactericidal effect of this invention. Although the targeted plant cell compound liquid has some auxiliary antimicrobial activity, it cannot replace the potent bactericidal function of the targeted antimicrobial peptide; Comparative Example 2 (lacking probiotic-PDRN superassembled nanoparticles), Comparative Example 3 (lacking PQQ-cyclodextrin inclusion complex), and Comparative Example 4 (without stacked microsphere controlled-release carrier) also showed similar effects. Comparative Example 5 (ordinary physical mixture replacing superassembly) and Comparative Example 6 (free PQQ replacing inclusion complex) showed inhibition zones that were comparable to those of Example 1 (difference ≤1.0 mm), which was as expected. This is because the targets of these components are bacterial colonization, mucosal repair and immune activation (in vivo action), and they do not directly contribute antibacterial activity in the Oxford Cup method without the participation of the immune system in vitro.

[0083] 2. Cell viability and mitochondrial function assay: Human vaginal epithelial cells VK2 / E6E7 were used to detect the effects of Examples 1-3 and Comparative Examples 1-6 on cell viability and mitochondrial ATP content. The results are as follows:

[0084]

[0085] The results showed that the enhancement of vaginal epithelial cell viability and mitochondrial ATP content by Examples 1-3 exhibited a clear concentration gradient dependence—the highly effective concentrated form (Example 2) enhanced cell viability to 146.3% and ATP content to 178.6%, demonstrating the most significant effect; the standard formula (Example 1) was second (128.5% / 152.0%); and the daily maintenance form (Example 3) was third (113.2% / 131.4%), all significantly better than the blank control group; Comparative Example 3 (lacking PQQ-cyclodextrin inclusion complex) showed only 101.5% cell viability (almost no enhancement) and only 103.2% ATP (almost no activation). The third group (Leonurus japonicus cyclodextrin enrichment solution + PQQ-cyclodextrin inclusion complex) showed the worst effect among all groups, demonstrating that the "nutrient supply + energy activation" pathway of the synergistic component (Leonurus japonicus cyclodextrin enrichment solution + PQQ-cyclodextrin inclusion complex) is the core driving force for enhanced mitochondrial function. PQQ, as an activator of mitochondrial biosynthesis, almost restored cellular energy metabolism to baseline levels after its absence. While Comparative Example 6 (free PQQ replacing PQQ-cyclodextrin inclusion complex) showed better cell viability (122.0%) and ATP (140.0%) than Comparative Example 3 (completely lacking PQQ), it was still significantly lower than Example 1 (128.5% / 152.0%), demonstrating the protective effect of cyclodextrin inclusion technology on PQQ. Stability is crucial – free PQQ partially degrades and becomes inactive in the culture medium, failing to fully exert its mitochondrial activation effect; in Comparative Example 2 (probiotic-deficient PDRN superassembled nanoparticles) and Comparative Example 5 (physical hybrid replacement superassembly), cell viability (118.5% / 118.0%) and ATP (132.0% / 132.5%) were lower than in Examples 1 and 3, but higher than in Comparative Example 6, falling into the intermediate level. This confirms that PDRN itself has inherent activities to promote cell proliferation and energy metabolism (free PDRN still has an effect), but the superassembled structure significantly enhances the delivery efficiency and cellular uptake of PDRN – at the same dosage, the superassembled structure… The effect of the loading (Example 1) was significantly better than that of physical mixing (Comparative Example 5); the cell viability (126.0%) and ATP (148.0%) of Comparative Example 1 (lacking the antimicrobial peptide MetaAMP12) were basically the same as those of Example 1, which was as expected—the target of the antimicrobial peptide is pathogenic bacteria rather than human epithelial cells, and its absence does not affect cell viability and mitochondrial function, proving that the functions of each component of the present invention are clearly defined and do not interfere with each other; the cell viability and ATP of Comparative Example 4 (without controlled-release carrier) were basically the same as those of Example 1, which was as expected—the in vitro cell experiment was a one-time administration, and the long-acting release mechanism of the sustained-release carrier did not play a role in short-term in vitro culture.

[0086] 3. Clinical Trial Results: 270 female subjects with vaginal flora imbalance and low immunity were recruited and randomly divided into 9 groups of 30 each. Each group used the products from Examples 1-3 and Comparative Examples 1-6, respectively, once daily for 7 consecutive days. The results are as follows:

[0087]

[0088] The results showed that Example 2 (high-efficacy concentrated form) had the best overall performance across all indicators, with a recovery rate of 96.7%, a symptom relief rate of 100%, and a recurrence rate of only 3.3% after 3 months. Its active ingredients (antimicrobial peptides, probiotic nanoparticles, PQQ inclusion complexes, repair peptides, etc.) were 1.5 to 2 times higher than those in Example 1, significantly enhancing the synergistic efficacy of sterilization, repair, and immune activation within a safe range, thus verifying the scientific validity of the "concentration-enhanced" principle of this invention. Example 1 (standard formula) showed excellent results, with a recovery rate of 93.3%, a relief rate of 96.7%, and a recurrence rate of 6%. 7% represents an economical option that balances efficacy and cost. Example 3 (daily maintenance type) showed a lower recovery rate (76.7%) and remission rate (83.3%) due to the lowest active ingredient concentration, with a relapse rate rising to 20.0%, but still outperformed all comparative examples, demonstrating that even at low doses, the multi-component synergy of this invention can still provide basic protection. Comparative Example 1 (lacking antimicrobial peptide MetaAMP12) had a recovery rate of only 43.3% and a relapse rate as high as 46.7%, confirming the "broad-spectrum bactericidal" effect of the first synergistic combination (targeted plant cell compound liquid + antimicrobial peptide). This is a prerequisite for initiating microecological reconstruction; without it, pathogenic bacteria cannot be effectively eliminated. Comparative Example 2 (lacking probiotics-PDRN superassembled nanoparticles) showed significantly lower recovery rates (60.0%) and remission rates (66.7%) than the Example 1, with a relapse rate of 33.3%. This indicates that the "inoculation and repair" function of the second synergistic group (probiotics-PDRN superassembly + transdermal repair peptides) was lost, leading to failed beneficial bacteria colonization and hindered mucosal repair. Comparative Example 3 (lacking PQQ-cyclodextrin inclusion complex) had the worst performance across all indicators (recovery rate 36.7%, relapse rate 56.7%), highlighting the importance of the third synergistic group (Leonurus japonicus iridoids + PQQ inclusion complex). "Immune activation" is the ultimate defense against relapse; without it, the body cannot actively resist the reinvasion of pathogens. Comparative Example 4 (lacking the stacked microsphere controlled-release carrier) showed a short-term recovery rate (90.0%) and remission rate (93.3%) close to Example 1, but the 3-month relapse rate soared to 40.0%, six times that of Example 1. This precisely demonstrates that although the controlled-release carrier does not participate in acute-phase bactericidal activity, it is a key technology for achieving long-term sustained release, maintaining local effective concentration, and preventing long-term relapse. Comparative Example 5 (ordinary physical mixture replacement) The recovery rate (66.7%), remission rate (73.3%), and relapse rate (30.0%) of the superassembled PDRN nanoparticles were all significantly lower than those in Example 1, demonstrating that the probiotic-PDRN superassembled nanoparticles are not simply a mixture. Only through the "bacterium-repair factor" supramolecular structure formed by hydrogen bonds and electrostatic attraction can the efficient loading and targeted delivery of PDRN be achieved, exerting a synergistic therapeutic effect of "one sphere, two effects." Comparative Example 6 (free PQQ replacing PQQ-cyclodextrin inclusion complex) had a recovery rate of only 50.0% and a relapse rate of 43%.The effect of 3% was even weaker than that of control group 2 (lacking probiotics). This is because free PQQ is extremely unstable in the formulation and vaginal environment, and is easily oxidized and inactivated. Cyclodextrin inclusion technology significantly improves the stability and bioavailability of PQQ, proving that this inclusion process is the core guarantee for the immune-activating effect of PQQ.

[0089] 120 volunteers with vaginal microecological imbalance (aged 22-55 years) were selected and randomly divided into 9 groups (20 participants in each of the Example 1-3 groups and 10 participants in each of the Comparative 1-6 groups). The treatment was administered continuously for 28 days. The results are as follows:

[0090]

[0091] The results showed that in the 28-day microecological reconstruction evaluation, Example 2 (high-efficacy concentrated type) had the best overall long-term indicators, with a pH recovery rate of 100% (all subjects returned to normal), a lactobacillus content as high as 92.5%, a mucosal repair score of 9.8 / 10, an sIgA increase rate of 78.5%, and symptoms disappearing in only 3.5 days. Its active ingredients (antimicrobial peptides, probiotic-PDRN superassembly, PQQ inclusion complex, repair peptides, etc.) were 1.5 to 2 times that of Example 1, significantly enhancing the three-level synergistic efficacy of "bactericidal-inoculative-protective" within a safe range, verifying the scientific nature of the concentration-enhanced efficacy of this invention; Example 1 (standard formula) had excellent effects (pH recovery 95%, lactobacillus 84.0%, sIgA increase 66.0%, symptoms disappearing in 5.3 days), making it an ideal solution that balances efficacy and cost; Example 3 (daily maintenance type) Because of the lowest concentration of active ingredients, all indicators (pH recovery 90%, lactobacillus 80.0%, sIgA increase 54.0%, symptom disappearance 6.2 days) were lower than those of Examples 1 and 2, but still better than all comparative examples, proving that even at low doses, the multi-component synergy can still play a basic protective role. Comparative Example 1 (lacking antimicrobial peptide MetaAMP12) had a pH recovery rate of only 70%, a lactobacillus percentage of only 62%, and required 10.5 days for symptom disappearance, confirming that the "precise sterilization" of the first synergistic group (targeted plant cell compound liquid + MetaAMP12) is a prerequisite for microecological reconstruction—if pathogenic bacteria are not eliminated, lactobacillus cannot colonize, and even with subsequent inoculation and protection components, they cannot play a role. Comparative Example 2 (lacking probiotics-PDRN super-assembled nanoparticles) showed a significant decrease in the lactobacillus percentage (72.0%) and sIgA increase rate (44.0%) compared to Example 1, indicating that the "inoculation and repair" of the second synergistic group (probiotics-PDRN super-assembled + transdermal repair peptide) is more effective. Without it, beneficial bacteria cannot firmly colonize the mucosal surface, and PDRN cannot be efficiently delivered to damaged tissues, hindering both deep repair and immune barrier reconstruction. Comparative Example 3 (lacking PQQ-cyclodextrin inclusion complex) had the worst performance in all indicators (sIgA increase of only 28.0%, lactobacilli only 54.0%, and symptom disappearance time as long as 11.5 days). This highlights that the third synergistic combination (Leonurus japonicus iridoids + PQQ inclusion complex) is the ultimate defense against the collapse of the microecology by protecting bacteria and activating immunity. Without PQQ to activate mitochondrial energy metabolism and immune pathways, the reconstructed microbiota cannot be maintained in the long term, and the body cannot actively resist the re-invasion of pathogens. Comparative Example 4 (lacking stacked microsphere controlled-release carrier) had the same pH recovery rate (90%) and lactobacillus percentage (80.0%) as Example 3, which seemed to have a good effect in the short term, but the sIgA increase rate (56.0%) was significantly lower than that of Example 1 (66.0%), and the symptom disappearance time was extended to 7 days.The 0-day timeframe demonstrates that while the controlled-release carrier does not participate in acute-phase bactericidal activity, its 12-24 hour graded sustained-release mechanism is a key technology for maintaining local effective drug concentration, continuously promoting sIgA secretion, and accelerating symptom resolution. Its absence significantly reduces long-term efficacy. In Example 5 (ordinary physical mixing replacing superassembly), all indicators (Lactobacillus 74.0%, sIgA 48.0%, symptom disappearance 8.3 days) were inferior to Example 3, proving that the probiotic-PDRN superassembly structure is not a simple physical mixture—only a supramolecular structure of "bacteria-repair factor" formed through hydrogen bonds and electrostatic attraction can achieve high-density PDRN loading (≥500,000 units). The cyclodextrin inclusion complex (PQQ) delivers PQQ to specific cells, achieving a synergistic therapeutic effect of "one sphere, two effects." While Comparative Example 6 (free PQQ replacing PQQ-cyclodextrin inclusion complex) showed better results in terms of Lactobacillus percentage (64.0%) and sIgA enhancement rate (38.0%) than Comparative Example 3 (54.0%, 28.0%) which completely lacked PQQ, it was significantly lower than Example 1 (84.0%, 66.0%). This demonstrates that free PQQ is easily oxidized and degraded in the formulation and vaginal environment, exhibiting only limited activity. The cyclodextrin inclusion complex significantly improves the stability and bioavailability of PQQ, serving as the core guarantee for achieving the third group's "bacterial protection" immune activation efficacy.

[0092] This invention employs a triple strategy of "bactericidal action (antimicrobial peptides + matrine) – probiotic inoculation (probiotic-PDRN superassembly) – probiotic protection (leonurus japonicus iridoids + PQQ inclusion complex)" supplemented by a layered microsphere controlled-release carrier to achieve long-lasting sustained release. It relies on two core technologies, superassembly and inclusion complex, to ensure stable and efficient delivery of the active ingredients. Experiments have shown that each component is indispensable, and the therapeutic effect is positively correlated with the concentration of the active ingredient within a safe range. The high-efficacy concentrated form (Example 2) is the optimal solution, suitable for subjects with severe infections or requiring intensive intervention; the standard formula (Example 1) is suitable for routine use; and the daily maintenance form (Example 3) can be used as a long-term maintenance option. This invention provides a graded, scientific, and comprehensive nursing solution for individuals with vaginal flora imbalance and weakened immunity.

[0093] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A product for improving vaginal flora balance and enhancing immunity, characterized in that, The product comprises the following components in parts by weight: Phase A: 65-80 parts water, 0.5-2 parts carbomer U20, 3-8 parts glycerol, 0.2-1 parts matrine; Phase B: 1-5 parts of directional plant cell compound solution, 1-5 parts of probiotic-PDRN super-assembled nanoparticles, 1-4 parts of Leonurus japonicus iridoid enrichment solution, 0.01-0.1 parts of PQQ-cyclodextrin inclusion complex, 1-5 parts of transdermal repair peptide, 1-5 parts of specific feminine hygiene antimicrobial peptide, and 1-5 parts of layered microsphere controlled-release carrier. The product comprises three synergistic components: Group 1: Targeted plant cell compound liquid and specific private care antimicrobial peptide, with a mass ratio of 1:0.5-1:2, synergistically achieve the dual effects of broad-spectrum bactericidal effect and beneficial bacteria promotion; The second group consists of probiotic-PDRN super-assembled nanoparticles and transdermal repair peptides, with a mass ratio of 1:0.5-1:2, which synergistically achieve deep mucosal repair and cell regeneration. The third group: Leonurus japonicus cyclohexene ether enrichment solution and PQQ-cyclodextrin inclusion complex, with a mass ratio of 1:0.005-1:0.05, synergistically achieves immune enhancement by improving microcirculation and activating mitochondria; The method for preparing the layered microsphere controlled-release carrier is as follows: the active ingredient is dissolved in an organic solvent to form an oil phase; in the presence of a surfactant, the oil phase is dispersed in an aqueous phase to form a primary emulsion; the active ingredient is self-assembled into a nanoscale layered microsphere structure by evaporating the organic solvent; the microspheres have a particle size of 50-500 nm and an encapsulation rate of not less than 85%; and sustained release is achieved in a vaginal environment of pH 3.8-4.5 for 12-24 hours. The specific feminine care antimicrobial peptide is MetaAMP12, an antimicrobial peptide with selective antibacterial activity, which has a killing effect on vaginal pathogens but no inhibition on lactobacilli. The preparation method of the probiotic-PDRN superassembled nanoparticles is as follows: Lactobacillus plantarum 299v is mixed with PDRN solution with a concentration of 0.1-5 mg / mL at a mass ratio of 1:50, and incubated at pH 6.8 and 35℃ for 3 hours. PDRN molecules are self-assembled and attached to the surface of the bacteria by hydrogen bonding and electrostatic attraction to form superassembled units with a particle size of 80-150 nm. Each unit is loaded with no less than 500,000 PDRN molecules. The nanoparticles are then lyophilized and reconstituted before use. The targeted plant cell compound solution is composed of extracts of Coptis chinensis, Phellodendron chinense, Sophora flavescens, Cnidium monnieri, Lonicera japonica, Forsythia suspensa, Leonurus japonicus, and Glycyrrhiza uralensis in a mass ratio of (1-2):(1-2):(2-3):(1-1.5):(1-2):(1-2):(1-1.5):(2-3), and its total flavonoid content is not less than 15wt%.

2. The product for improving vaginal flora balance and enhancing immunity according to claim 1, characterized in that, The specific feminine antimicrobial peptide has a minimum inhibitory concentration (MIC) of ≤8 μg / mL against Gardnerella vaginalis and a half-maximal inhibitory concentration (IC50) of ≥128 μg / mL against Lactobacillus.

3. The product for improving vaginal flora balance and enhancing immunity according to claim 1, characterized in that, The transdermal repair peptide is palmitoyl pentapeptide-4 or acetyl hexapeptide-8, which achieves targeted penetration into the deep folds of the vaginal mucosa by coupling with hyaluronic acid modified on the surface of a stacked microsphere controlled-release carrier.

4. The product for improving vaginal flora balance and enhancing immunity according to claim 1, characterized in that, The preparation process of the PQQ-cyclodextrin inclusion complex is as follows: PQQ and β-cyclodextrin are stirred in a water bath at 60°C for 2 hours at a molar ratio of 1:1.2, and then freeze-dried to obtain the inclusion complex. Its stability in a pH 4.0-5.0 environment is more than 3 times higher than that of free PQQ.

5. The product for improving vaginal flora balance and enhancing immunity according to claim 1, characterized in that, The final pH value of the product is adjusted to 3.8-4.5, the osmotic pressure is 260-320 mOsm / kg, it is isotonic with the physiological environment of the vagina, and it does not contain any artificial preservatives.

6. A method for preparing a product for improving vaginal flora balance and enhancing immunity as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Add the purified water required for phase A to the vacuum emulsifier, add carbomer U20, glycerol and matrine, heat to 85°C at a rate of 2°C / min, turn on the homogenizer and shear at 5000rpm for 15 minutes, and sterilize at a constant temperature for 10 minutes. S2: When the temperature drops to 50℃, add the directional plant cell compound solution, the motherwort iridoid enrichment solution, and the PQQ-cyclodextrin inclusion complex in sequence, and stir at 200 rpm to dissolve. S3: When the temperature drops to 42℃, slowly add the probiotic-PDRN super-assembled nanoparticles, transdermal repair peptides, specific feminine care antimicrobial peptides and layered microsphere controlled-release carrier suspension to the system, and control the stirring speed to ≤300rpm. S4: Adjust the pH to 4.0 with lactic acid, test the viscosity and microbial limits, and after passing the test, filter through a 0.22μm microporous membrane for sterilization and then fill.

7. The method for preparing the product for improving vaginal flora balance and enhancing immunity according to claim 6, characterized in that, The method of adding the layered microsphere controlled-release carrier suspension in step S3 is as follows: a microfluidic injection pump is used to inject at a constant rate of 0.5 mL / min, and an ultrasonic probe (frequency 40 kHz) is set at the injection port for online dispersion to prevent microsphere aggregation.

8. The product for improving vaginal flora balance and enhancing immunity according to claim 1, characterized in that, The product is suitable for at least one of the following symptoms: vulvar itching, abnormal vaginal discharge, vaginal microecological imbalance, and cervicitis.

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