Plant composite antibacterial liquid composition for human papilloma virus infection and preparation method thereof

Abstract

The invention discloses a plant composite antibacterial liquid composition aiming at HPV (human papillomavirus) infection and a preparation method thereof, and belongs to the technical field of biological medicines. The composition is prepared from the following components in parts by weight: 5 to 20 parts of brucea javanica extract, 3 to 15 parts of senna extract, 2 to 10 parts of anhydridized beta-lactoglobulin, 1 to 8 parts of dandelion extract, 1 to 5 parts of penetration enhancer, 0.1 to 1 part of pH (Potential of Hydrogen) regulator, 0.1 to 0.5 part of preservative and the balance of phosphate buffer solution. According to the invention, anhydridized beta-lactoglobulin is innovatively taken as a carrier, so that targeted delivery of active ingredients is realized; the anti-HPV effect is enhanced through the synergistic effect of the brucea javanica and the kalii extract; the anti-inflammatory repair function of the dandelion extract is combined, and a compound penetration enhancer and a specific preparation process are matched, so that the product has the characteristics of efficient HPV resistance, anti-inflammatory repair, mildness, no irritation and good stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a plant-based compound antibacterial liquid composition for human papillomavirus (HPV) infection and its preparation method. Background Technology

[0002] Human papillomavirus (HPV) is a non-enveloped, double-stranded circular DNA virus. Its viral particles consist of capsid proteins (L1, L2) and core DNA, and can specifically infect human skin and mucous membrane epithelial cells. Based on their carcinogenicity, HPV can be divided into low-risk types (such as HPV 6 and 11, mainly causing benign lesions like common warts, flat warts, and condyloma acuminata) and high-risk types (such as HPV 16, 18, 31, and 33, persistent infection of which can lead to malignant tumors such as cervical cancer, anal cancer, and vulvar cancer). Persistent infection with high-risk HPV is the leading cause of cervical cancer—more than 500,000 people die globally each year from HPV-related diseases, with cervical cancer accounting for over 80% of these deaths, seriously threatening human life and health.

[0003] Currently, clinical intervention methods for HPV infection are mainly divided into three categories: Physical therapy, including laser therapy, cryotherapy, electrocautery, and microwave therapy, uses physical energy to destroy HPV-infected lesions (such as warts), achieving the goal of "removing the lesions." The advantages of this method are rapid onset of action and direct lesion removal, but it has significant drawbacks: First, it is highly invasive, easily causing skin and mucous membrane damage and bleeding after treatment, and may leave scars (especially on exposed areas such as the face and neck); second, it has a high recurrence rate, as physical therapy can only remove visible lesions and cannot eliminate HPV viruses latent in normal epithelial cells, with a clinical recurrence rate of 30%-50%; third, its applicability is limited, as it is difficult to precisely target lesions in hidden areas such as the cervical canal and deep within the vagina.

[0004] Chemotherapy: Commonly used drugs include fluorouracil ointment (a cytotoxic drug that inhibits the proliferation of diseased cells), imiquimod cream (an immunomodulator that activates the local immune response to clear the virus), and interferon gel (a broad-spectrum antiviral drug that inhibits viral replication). Although this method can act on the lesion through topical application, it has the following problems: First, it is highly irritating. Cytotoxic drugs such as fluorouracil also have a killing effect on normal skin and mucous membrane cells, easily leading to local redness, swelling, pain, and erosion. Second, there is a risk of drug resistance. Long-term use of a single chemical drug can easily lead to drug-resistant mutations in the HPV virus, reducing the treatment effect. Third, the efficacy is limited. Most chemical drugs can only target a single aspect of "viral replication" or "disease cell proliferation," and cannot simultaneously address the comprehensive needs of "virus clearance + inflammation reduction + mucosal repair."

[0005] Immunotherapy includes HPV vaccines (preventative, such as bivalent, quadrivalent, and nonavalent vaccines, which can prevent infection with specific HPV subtypes but cannot clear existing infections) and adoptive immunotherapy (such as CTL cell therapy, which works by infusing immune cells that specifically kill HPV-infected cells). The core of this approach is to clear the virus by regulating the body's immune function, but it has limitations: First, preventative vaccines cannot cover all HPV subtypes (currently, the nonavalent vaccine only covers 9 subtypes, while there are over 200 known HPV subtypes), and they are ineffective against already infected individuals; second, therapeutic immunotherapy (such as CTL cell therapy) is slow to take effect (usually requiring 2-3 treatment cycles, each lasting 1-2 months), has a long treatment period, and is expensive (a single treatment can cost over 10,000 yuan), making it difficult to popularize in primary healthcare institutions.

[0006] Plant extracts, due to their advantages such as natural origin, good biocompatibility, few side effects, and multiple targets, are gradually becoming a research hotspot in the field of antiviral therapy. Existing research shows that: However, the application of plant extracts in HPV infection intervention in existing technologies has significant shortcomings: Lack of synergistic effect: Most existing products are single plant extracts or simple compound formulations (such as a mixture of Brucea javanica and dandelion extracts). The proportions of the ingredients have not been optimized through experimental screening, so they cannot achieve the synergistic effect of "antiviral + anti-inflammatory + repair", resulting in low HPV inhibition rate (usually below 70%) and slow wound healing.

[0007] Low delivery efficiency: The active ingredients in plant extracts (such as crocin and mancacaridine) are mostly lipid-soluble or weakly water-soluble, with poor skin and mucous membrane permeability and are easily degraded by local enzymes, resulting in insufficient concentration of active ingredients at the site of HPV infection, which cannot effectively eliminate latent viruses.

[0008] Poor stability: Polyphenols and terpenoids in plant extracts are easily oxidized and hydrolyzed, and may interact with other components (such as preservatives and buffers), leading to degradation of active ingredients and stratification of appearance during product storage, with a shelf life of usually less than 6 months.

[0009] Irritation risk: Some plant extracts (such as crocin) have certain cytotoxicity. Direct application to broken skin and mucous membranes can easily lead to local irritation reactions (such as redness, swelling, and itching). Furthermore, existing products have not been optimized for the pH value (5.5-7.5) of skin and mucous membranes, which further increases the risk of irritation.

[0010] Anhydride-modified β-lactoglobulin is a whey protein modified with maleic anhydride, possessing the following characteristics: First, it has good biocompatibility, derived from milk protein, is non-immunogenic, and can be naturally metabolized by the human body; second, it has strong mucosal adhesion, as the anhydride-modified molecule carries a negative charge on its surface, allowing it to bind to positively charged groups (such as amino groups) on the surface of skin and mucous membrane epithelial cells through electrostatic interactions, prolonging its residence time on the mucosal surface; third, it has strong drug encapsulation capabilities, encapsulating lipid-soluble active ingredients through hydrophobic interactions and hydrogen bonding to form nanoscale carrier particles (0.1-0.5 μm in diameter), protecting the active ingredients from degradation. However, there are currently no reports on the use of anhydride-modified β-lactoglobulin in combination with extracts of *Brucea javanica*, *Cannabis sativa*, and *Taraxacum mongolicum* for HPV infection intervention. Furthermore, how to precisely control the degree of anhydride modification (avoiding excessive toxicity due to high levels and insufficient carrier function due to low levels) and optimize the preparation process (avoiding degradation of active ingredients and achieving uniform dispersion of components) remains a pressing technical challenge.

[0011] Therefore, developing a plant-based compound antibacterial liquid composition with good stability and mild, non-irritating properties, based on an integrated design of "targeted delivery + synergistic antiviral + anti-inflammatory repair", is of great clinical significance and application value for addressing the shortcomings of existing HPV infection intervention products and improving clinical nursing outcomes. Summary of the Invention

[0012] This invention provides a plant-based compound antibacterial liquid composition for HPV infection, particularly suitable for the prevention and adjunctive intervention of skin and mucous membrane lesions (such as common warts, flat warts, and condyloma acuminata) caused by HPV infection. It can be widely applied in clinical nursing scenarios such as gynecology (e.g., adjunctive care for cervical HPV infection) and dermatology (e.g., care for skin wart lesions), as well as in daily skin and mucous membrane HPV infection prevention and health care scenarios. By weight, it comprises the following components: 5-20 parts of Brucea javanica extract, 3-15 parts of Manjacaranda extract, 2-10 parts of anhydride-modified β-lactoglobulin, 1-8 parts of Taraxacum mongolicum extract, 1-5 parts of penetration enhancer, 0.1-1 part of pH adjuster, 0.1-0.5 parts of preservative, with the balance being phosphate buffer.

[0013] Furthermore, a method for preparing a plant-based compound antibacterial liquid composition targeting HPV infection is also provided, comprising the following steps: Carrier solution preparation: Take 2-10 parts of anhydride-modified β-lactoglobulin, add 500-600 parts of phosphate buffer, stir at 30-35℃ and 200-250 rpm for 15-20 min until dissolved to obtain the carrier solution; Plant extract compound: Take 5-20 parts of Brucea javanica extract, 3-15 parts of Manjacali extract, and 1-8 parts of Taraxacum mongolicum extract, add them to the carrier solution, and stir at 300-350 rpm for 30-40 min to obtain mixture A; Adding a penetration enhancer: Add 1-5 parts of a penetration enhancer to mixture A, stir at 250-300 rpm for 15-20 minutes to obtain mixture B; pH adjustment: Add 0.1-1 part of pH adjuster to mixture B, stir at 150-200 rpm to adjust the pH to 6.5-7.5, to obtain mixture C; Preservative: Add 0.1-0.5 parts of preservative to mixture C, stir at 150-200 rpm for 5-10 minutes to obtain mixture D; Solvent replenishment and pre-filtration: Add the remaining phosphate buffer to mixture D to a total weight of 1000 parts, stir, and then filter through a 0.45 μm filter membrane to obtain the pre-filtered solution; High-pressure homogenization: Homogenize the pre-filtered liquid 2-3 times under a pressure of 30-35 MPa, each time for 5-8 minutes, to obtain a homogenized liquid; Fine filtration and filling: The homogenized liquid is filtered through a 0.22μm sterile filter membrane, filled into light-proof pharmaceutical bottles under sterile conditions, and sealed.

[0014] Furthermore, the extract of Brucea javanica is prepared by reflux extraction with an ethanol-water mixed solvent, the volume ratio of ethanol to water is 7:3, the extraction temperature is 70-80℃, the extraction time is 2-3h, and after purification with macroporous resin, the content of crotonin is ≥10%.

[0015] Furthermore, the mangacali extract is prepared by supercritical CO2 extraction at an extraction temperature of 35-45℃ and an extraction pressure of 25-35MPa. The entrainer is a 10% (v / v) aqueous ethanol solution, and the mangacali glycoside content in the extract is ≥8%.

[0016] Furthermore, the preparation method of the anhydride-modified β-lactoglobulin is as follows: β-lactoglobulin is dissolved in an acetate-sodium acetate buffer solution at pH 6.0-6.5, maleic anhydride is added, the mass ratio of β-lactoglobulin to maleic anhydride is 10:1-15:1, the reaction is carried out at 30-35℃ for 2-3 hours, and after dialyzing, it is freeze-dried to obtain the product, which has an anhydride degree of 20-30%.

[0017] Furthermore, the dandelion extract is prepared by water decoction-ethanol precipitation method. Water is added and decocted twice, each time for 1.5-2 hours. The decoctions are combined, concentrated, and then ethanol is added to a volume fraction of 70%. The mixture is allowed to stand and precipitate. The supernatant is then concentrated and dried, and the chlorogenic acid content is ≥5%.

[0018] Furthermore, the penetration enhancer is a compound of azone and propylene glycol in a weight ratio of 1:3 to 1:5.

[0019] Furthermore, the pH value of the phosphate buffer solution is 7.2-7.4, and the pH value of the composition is 6.5-7.5.

[0020] Furthermore, the pH adjuster is selected from at least one of sodium hydroxide and hydrochloric acid; the preservative is selected from at least one of phenoxyethanol and ethylparaben.

[0021] Furthermore, the high-pressure homogenization temperature in step 7) is 30-35℃; the light-proof medicine bottle in step 8) is a brown glass bottle, and the filling environment is a Class 100 sterile clean room.

[0022] Beneficial effects: The core innovation lies in breaking through existing technological bottlenecks and constructing an integrated plant-based compound antibacterial liquid system that combines "targeted delivery, synergistic antiviral activity, anti-inflammatory repair, and stable compatibility," specifically reflected in the following eight aspects: This invention innovatively utilizes anhydride-mediated β-lactoglobulin as a carrier of active ingredients, achieving triple functionality through precise control of the anhydride degree (20-30%): Targeted binding: The carboxyl group (-COOH) on the surface of the anhydride-treated protein can specifically bind to the amino group (-NH2) on the surface of the HPV viral capsid protein (L1) through electrostatic interaction, allowing the carrier-drug complex to be targeted and enriched at the HPV infection site. Mucosal adhesion: Anhydride-modified β-lactoglobulin can bind to mucins on the surface of skin and mucosal epithelial cells through hydrogen bonding, prolonging its residence time on the mucosal surface (4-6 hours longer than that of the free active ingredient). Drug protection: Lipid-soluble active ingredients (such as crocin) are encapsulated through hydrophobic interactions, preventing their degradation by local enzymes (such as esterases). Experiments show that this delivery system increases the concentration of active ingredients at the HPV infection site by 3-5 times compared to free components, significantly enhancing the anti-HPV effect.

[0023] The synergistic anti-HPV mechanism of Brucea javanica and Manjakari extracts was investigated through orthogonal experiments to screen the optimal ratio of Brucea javanica extract (5-20 parts) to Manjakari extract (3-15 parts) to achieve synergistic "dual-target" action. Brucea javanica extract contains crotonin, which can inhibit the activity of HPV viral DNA polymerase (E1 protein) and prevent viral DNA replication. Mangacalin in mangacalin extract can bind to HPV capsid protein (L1), disrupting the integrity of the capsid structure, exposing the viral core DNA, and allowing it to be degraded by the host nucleases.

[0024] Experiments have shown that this synergistic system can inhibit HPV types 16 and 18 high-risk viruses by more than 90%, which is significantly better than single extracts (the inhibition rate of single Brucea javanica extract is 62-68%, and the inhibition rate of single Manjacali extract is 58-65%).

[0025] The anti-inflammatory and repairing synergistic effects of dandelion extract: The introduction of dandelion extract (1-8 parts) not only compensates for the deficiency of existing products that "only have antiviral properties but lack anti-inflammatory and repairing effects," but also forms a synergistic effect with anti-HPV ingredients: Anti-inflammatory effect: Chlorogenic acid in dandelion extract can inhibit the expression of inflammatory factors (IL-1β, TNF-α) caused by HPV infection and reduce local inflammatory response (the level of inflammatory factors is reduced by 40-50% compared with the control group); Mucosal repair effect: Chlorogenic acid can promote the proliferation and migration of skin and mucosal epithelial cells (such as keratinocytes and cervical epithelial cells), and accelerate the healing of damaged mucosa.

[0026] Clinical simulation experiments show that after using this composition, the healing time of genital warts caused by HPV infection is shortened by 2-3 days compared with the control group (without dandelion extract), and the mucosal repair rate is increased by more than 40%.

[0027] Differentiated extraction and purification processes for plant extracts are designed specifically for the characteristics of different plant components, ensuring stable levels of active ingredients and minimal impurities. Brucea javanica: Extracted by reflux of ethanol-water (7:3) + purified by macroporous resin to avoid water-soluble impurities (such as polysaccharides) residue and ensure that the content of crotonin is ≥10%; Mangacali: Supercritical CO2 extraction (with 10% ethanol entrainer) and low-temperature extraction (35-45℃) avoid degradation of heat-sensitive components (mangacali glycosides), resulting in a 30% higher yield than traditional ethanol extraction; Dandelion: The water decoction-alcohol precipitation method (70% ethanol precipitation) is used to remove impurities such as protein and starch, while retaining water-soluble active ingredients such as chlorogenic acid, with a content of ≥5%.

[0028] This process ensures that the batch-to-batch variation of active ingredients in each extract is ≤5%, guaranteeing consistent product efficacy.

[0029] The mucosal penetration enhancement technology using a compound penetration enhancer combines azone and propylene glycol (1:3-1:5) to address the problem of poor mucosal permeability of plant extracts. Azone can disrupt the lipid bilayer structure of the stratum corneum of the skin and mucous membranes, forming "temporary channels" and increasing the permeability of the stratum corneum; Propylene glycol can increase the solubility of active ingredients (such as crotonin), reduce the retention of active ingredients in the stratum corneum, and promote their diffusion into the dermis or submucosal layer. The synergistic effect of the two increases the mucosal penetration of the active ingredient by 2-3 times compared to azone alone, ensuring that the drug concentration in deep HPV infection sites (such as 2-3 mm inside the cervical canal) reaches an effective inhibitory concentration (≥1 μg / mL).

[0030] Precise control of the degree of anhydrideation of β-lactoglobulin was achieved by adjusting the mass ratio of β-lactoglobulin to maleic anhydride (10:1-15:1) and reaction conditions (30-35℃, 2-3h), maintaining the degree of anhydrideation at 20-30% and balancing carrier function and safety. Low anhydride content (<20%): Insufficient number of carboxyl groups on the protein surface, weak mucosal adhesion and drug encapsulation ability, and low delivery efficiency of active ingredients; Excessive anhydride content (>30%): Increased risk of protein denaturation, and excessive surface carboxyl groups lead to a decrease in local pH, irritating the skin and mucous membranes (irritation response integral >0.5). When the degree of anhydride concentration is 20-30%, the mucosal adhesion rate of the carrier is ≥80%, the drug encapsulation rate is ≥85%, and the skin irritation response score is ≤0.3 (no irritation), resulting in the best overall performance.

[0031] pH compatibility optimization of the buffer system was achieved using a combination of "phosphate buffer (pH 7.2-7.4) + pH adjuster" to achieve dual optimization: Solvent compatibility: Phosphate buffer has good compatibility with acid-anhydrated β-lactoglobulin and plant extracts, with no precipitation or stratification. pH matching: The pH of the composition is adjusted to 6.5-7.5 by a pH adjuster, which is highly matched with the pH of human skin and mucous membranes (5.5-7.5), significantly reducing irritation.

[0032] Skin irritation tests (according to the 2015 edition of the Cosmetic Safety Technical Specifications) show that the average irritation response score of this composition is ≤0.3, which is a non-irritating product and is superior to existing similar products (average irritation response score 0.8-1.2, mild irritation).

[0033] The stability assurance process of high-pressure homogenization and aseptic filling introduces high-pressure homogenization (30-35MPa) and aseptic filling processes to solve the problem of poor product storage stability: High-pressure homogenization: This process forms a nano-dispersion system with uniform particle size (0.1-0.5μm) for the carrier-drug complex, preventing stratification or precipitation during storage (no stratification after 18 months of refrigerated storage at 4℃). 0.22μm fine filtration + aseptic filling: removes microorganisms and impurity particles, prevents secondary contamination during the filling process, and ensures product sterility (100% sterility inspection pass rate).

[0034] Stability tests show that this composition has a shelf life of up to 18 months under refrigeration at 4°C and up to 12 months under room temperature at 25°C, with an active ingredient retention rate of ≥85%, which is far superior to existing plant extract products (shelf life of 6-8 months, active ingredient retention rate ≤70%). Detailed Implementation Example 1 A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 12 parts (Brucea javanica content 12%) Manjacali extract: 8 parts (manjacali glycoside content 9%) Anhydride-modified β-lactoglobulin: 5 parts (25% anhydride modification) Dandelion extract: 4 portions (chlorogenic acid content 6%) Penetration enhancer: 4 parts (1 part azone + 3 parts propylene glycol, weight ratio 1:3) Preservative: 0.3 parts (phenoxyethanol) pH adjuster: appropriate amount (0.1 mol / L sodium hydroxide solution, adjust pH to 7.0) Phosphate buffer: Balance (make up to 1000 samples, pH 7.3) Its preparation method includes the following steps: Carrier solution preparation: Take 5 parts of anhydride-modified β-lactoglobulin, add 600 parts of phosphate buffer (pH 7.3), stir at 220 rpm for 18 min in a 32℃ constant temperature water bath until completely dissolved to obtain the carrier solution; Plant extract compound: Take 12 parts of Brucea javanica extract, 8 parts of Manjacali extract and 4 parts of Taraxacum mongolicum extract, mix them evenly and add the carrier solution. Stir at 320 rpm for 35 min at 32℃ to obtain mixture A. Addition of penetration enhancer: Take 1 part azone and 3 parts propylene glycol, mix them evenly, add them to mixture A, and stir at 280 rpm for 18 minutes at 32°C to obtain mixture B; pH adjustment: Slowly add 0.1 mol / L sodium hydroxide solution to mixture B, stir at 180 rpm until the pH value stabilizes at 7.0, to obtain mixture C; Preservation stage: Add 0.3 parts of phenoxyethanol to mixture C, stir at 180 rpm for 8 minutes to obtain mixture D; Solvent replenishment and pre-filtration: Add the remaining phosphate buffer to mixture D (to bring the total weight to 1000 parts), stir for 8 min, and then pre-filter through a 0.45 μm polyethersulfone membrane to obtain the pre-filtered solution; High-pressure homogenization: The pre-filtered liquid is fed into a high-pressure homogenizer and homogenized twice at 32 MPa and 32°C for 6 minutes each time to obtain a homogenized liquid; Fine filtration and filling: The homogenized solution is finely filtered through a 0.22μm sterile filter membrane, filled into 10mL light-proof pharmaceutical bottles in a Class 100 clean room, and sealed.

[0035] Example 2 A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 5 parts (10% crotonin content) Manjacali extract: 3 portions (manjacali glycoside content 8%) Anhydride-modified β-lactoglobulin: 2 portions (20% anhydride modification) Dandelion extract: 1 part (chlorogenic acid content 5%) Penetration enhancer: 0.8 parts (0.2 parts azone + 0.6 parts propylene glycol, weight ratio 1:3) Preservative: 0.1 parts (ethylparaben) pH adjuster: appropriate amount (0.1 mol / L sodium hydroxide solution, adjust pH to 6.5) Phosphate buffer: Balance (make up to 1000 samples, pH 7.2) Its preparation method is basically the same as that in Example 1, except that: Carrier solution preparation: temperature 30℃, stirring speed 200 rpm, stirring time 15 min; High-pressure homogenization: pressure 30MPa, homogenize twice, 5min each time; The target pH value is 6.5. The preservative ethylparaben must first be dissolved in 1 part propylene glycol before being added to mixture C.

[0036] Example 3 A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 20 parts (15% crotonin content) Manjacali extract: 15 parts (10% manjacali glycoside content) Anhydride-modified β-lactoglobulin: 10 parts (anhydride degree 30%) Dandelion extract: 8 parts (chlorogenic acid content 8%) Penetration enhancer: 6 parts (1.2 parts azone + 4.8 parts propylene glycol, weight ratio 1:4) Preservative: 0.5 parts (0.3 parts phenoxyethanol + 0.2 parts ethylparaben) pH adjuster: appropriate amount (0.1 mol / L hydrochloric acid solution, adjust pH to 7.5) Phosphate buffer: Balance (make up to 1000 samples, pH 7.4) Its preparation method is basically the same as that in Example 1, except that: Carrier solution preparation: temperature 35℃, stirring speed 250 rpm, stirring time 20 min; Plant extract blend: Stirring speed 350 rpm, stirring time 4 minutes continue 0min; High-pressure homogenization: pressure 35MPa, homogenization 3 times, 8min each time; The target pH value is 7.5.

[0037] Comparative Example 1 (without anhydride-modified β-lactoglobulin) An anti-HPV antibacterial liquid, which differs from Example 1 in that it does not contain anhydride-modified β-lactoglobulin, while the remaining components (parts by weight, preparation method) are the same as in Example 1.

[0038] Comparative Example 2 (without Mangacalis extract) An anti-HPV antibacterial liquid differs from that of Example 1 in that it does not contain mangacali extract, while the remaining components (parts by weight, preparation method) are the same as those in Example 1.

[0039] Comparative Example 3 (Single Permeability Enhancer) An anti-HPV antibacterial liquid differs from that of Example 1 in that the penetration enhancer is a single azone (4 parts), while the remaining components (parts by weight, preparation method) are the same as those in Example 1.

[0040] Performance testing To verify the technical effects of the present invention, the following performance tests were conducted on Examples 1-3 and Comparative Examples 1-3: 1. HPV Inhibition Effect Test Test Model: A HeLa cell model infected with HPV16 was used (HeLa cells are HPV16-positive cervical cancer cells). Test Method: Samples from Examples 1-3 and Comparative Examples 1-2 were diluted to 100 μg / mL and applied to HeLa cells (1×10⁻⁶ cells / mL). 5 Cells were cultured in a 37℃, 5% CO2 incubator for 48 h. The relative expression level of HPV16 E6 gene in cells was detected by real-time quantitative PCR (qPCR), and the viral inhibition rate was calculated (inhibition rate = (1 - relative expression level of E6 gene in experimental group / blank control group) × 100%). The test results are shown in Table 1 below.

[0041]

[0042] Results Analysis: Examples 1-3 all showed inhibition rates of over 88% against HPV16 virus, with Examples 1 and 3 exceeding 92%, indicating that the synergistic system of the present invention has excellent anti-HPV effect; Comparative Example 1, lacking anhydride-modified β-lactoglobulin carrier, could not target the delivery of active ingredients, resulting in an inhibition rate of only 65.1%; Comparative Example 2, lacking mangacalis extract, could not destroy the viral capsid, reducing the inhibition rate to 70.2%, confirming the key role of carrier technology and synergistic formulation.

[0043] 2. Mucosal repair effect test Test Model: SPF-grade SD rats (200-220g) HPV infection simulated skin and mucous membrane injury model (constructed by sanding the rat vaginal mucosa with HPV16 virus suspension). Test Method: Thirty model rats were randomly divided into 5 groups (n=6 per group). Samples from Examples 1-3 and Comparative Examples 1-2 were applied to each group (0.2mL twice daily for 7 consecutive days). The average healing time of the vaginal mucosal wounds in each group was observed and recorded. Test Results: As shown in Table 2 below.

[0044]

[0045] Results analysis: The samples in Examples 1-3 significantly shortened the mucosal wound healing time (4.1-4.5 days), thanks to the anti-inflammatory and repairing effects of dandelion extract and the mucosal protective effect of anhydride-modified β-lactoglobulin; the healing time of Comparative Example 1 (without carrier) and Comparative Example 2 (without mangacali) both exceeded 6.5 days, indicating that the synergistic effect of the components of the present invention can effectively promote mucosal repair.

[0046] 3. Skin irritation test Test Method: Referring to the repeated skin irritation test method in the "Cosmetic Safety Technical Specifications" (2015 edition), eight healthy New Zealand white rabbits (weighing 2.0-2.5 kg) were selected. After hair removal on their backs (hair removal area 5cm×5cm), the samples from Examples 1-3 (0.5 mL / rabbit) were applied to the left side, and physiological saline (negative control) was applied to the right side. This was done once daily for 14 consecutive days. Skin reactions (erythema, edema) were observed daily and scored according to the standard (erythema: 0-4 points; edema: 0-4 points), and the average irritation response score was calculated. Test Results: As shown in Table 3 below.

[0047]

[0048] Results analysis: The average irritation response scores of Examples 1-3 were all ≤0.3, which met the "non-irritating" evaluation criteria, indicating that the pH optimization and mild formulation design of the present invention can effectively reduce skin and mucous membrane irritation and is suitable for long-term local use.

[0049] 4. Stability Testing Test conditions: Samples from Examples 1-3 were stored under the following conditions: refrigerated at 4°C (protected from light), room temperature at 25°C (protected from light), and accelerated at 40°C (protected from light). Samples were taken and tested at 0 months, 3 months, 6 months, and 12 months, respectively. Test indicators: appearance (whether there was layering or turbidity), pH change (difference from the initial value), and retention rate of active ingredients (total amount of crotonin, mancacariin, and chlorogenic acid, detected by HPLC). Test results: As shown in Table 4 below (using 12-month data as an example).

[0050]

[0051] Results Analysis: After 12 months of storage at 4℃, the active ingredient retention rate of Examples 1-3 was still about 90%; after 12 months of storage at 25℃, the appearance and pH value changed little, and the active ingredient retention rate exceeded 87%, demonstrating excellent stability and meeting the shelf life requirements of 12-18 months in practical applications.

[0052] 5. Mucosal penetration test Test Model: Isolated vaginal mucosa from rats (taken from SPF-grade SD rats, approximately 0.3 mm thick). Test Method: A Franz diffusion cell was used (5 mL supply cell, 10 mL receiving cell, stirring speed 300 rpm, temperature 37°C). Samples from Example 1 and Comparative Example 3 (containing equal amounts of crotonin) were added to the supply cell, and phosphate buffer (pH 7.4) was added to the receiving cell. Samples were taken at 1 h, 2 h, 4 h, and 8 h, and the crotonin content in the receiving cell was detected by HPLC. The cumulative permeation was calculated. Test Results: At 8 h, the cumulative permeation of crotonin in Example 1 was (28.6 ± 2.3) μg / cm³. 2 Comparative Example 3 (single azone) had a concentration of (10.2 ± 1.5) μg / cm³. 2 This indicates that the compound penetration enhancer of the present invention can significantly enhance the mucosal penetration ability of the active ingredient (by 2.8 times).

[0053] The plant-based compound antibacterial liquid composition for HPV infection and its preparation method of the present invention achieve the following beneficial effects through multi-dimensional innovative design: Significant anti-HPV effect: The targeted delivery of the anhydride-modified β-lactoglobulin carrier, combined with the synergistic antiviral mechanism of Brucea javanica and Manjacali extract, can achieve an inhibition rate of over 90% against high-risk HPV types (such as types 16 and 18), solving the problem of low inhibition rate of existing plant extract products; Synergistic anti-inflammatory and repair functions: Dandelion extract exerts both anti-inflammatory (reducing the level of inflammatory factors) and mucosal repair (promoting the proliferation of epithelial cells) effects, shortening the healing time of HPV-infected wounds by 2-3 days and solving the problem of slow wound healing after physical therapy; Excellent biocompatibility: Through pH matching (6.5-7.5) with skin and mucous membranes and precise control of acid anhydride degree (20-30%), the product has an average irritation response score of ≤0.3, making it a non-irritating product suitable for sensitive mucous membrane areas such as the cervix and vagina. High stability and safety: High-pressure homogenization process ensures uniform particle size (0.1-0.5μm), aseptic filling ensures microbial control, shelf life can reach 12 months at room temperature of 25℃, and the retention rate of active ingredients is ≥87%, which solves the problems of easy degradation and short shelf life of plant extract products. It has a wide range of applications: it can be used as an adjunct treatment for skin lesions such as common warts, flat warts, and condyloma acuminata caused by HPV infection, as well as for daily care of cervical HPV infection. It has both preventive and intervention functions and is suitable for clinical use in gynecology and dermatology as well as for family health care.

[0054] Example 4 (using chitosan nanoparticle carrier) A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 10 parts (Brucea javanica content 11%) Cnidium monnieri extract: 6 parts (ostrichin content 8%) Chitosan nanoparticles: 8 parts (particle size 150-200nm, degree of deacetylation ≥90%) Viola yedoensis extract: 3 parts (6% luteolin content) Penetration enhancer: 3 parts (0.5 parts menthol + 2.5 parts propylene glycol, weight ratio 1:5) Preservative: 0.2 parts (methylparaben) pH adjuster: appropriate amount (0.1 mol / L hydrochloric acid solution, adjust pH to 6.8) Acetic acid-sodium acetate buffer: Balance (make up to 1000 parts, pH 6.5) Its preparation method includes the following steps: Carrier solution preparation: Take 8 parts of chitosan nanoparticles, add 550 parts of acetate-sodium acetate buffer (pH 6.5), stir at 230 rpm for 20 min in a constant temperature water bath at 28℃ until completely dispersed to obtain the carrier solution; Plant extract compound: Take 10 parts of Brucea javanica extract, 6 parts of Cnidium monnieri extract, and 3 parts of Viola yedoensis extract, mix them evenly, add the carrier solution, and stir at 340 rpm for 32 min at 33℃ to obtain mixture A; Addition of penetration enhancer: Take 0.5 parts of menthol and 2.5 parts of propylene glycol, heat and stir at 50°C until the menthol is completely dissolved, cool and add mixture A, stir at 270 rpm for 16 min at 33°C to obtain mixture B; pH adjustment: Slowly add 0.1 mol / L hydrochloric acid solution to mixture B, stir at 170 rpm until the pH value stabilizes at 6.8, to obtain mixture C; Preservative stage: Add 0.2 parts of methylparaben to mixture C, stir at 170 rpm for 7 min to obtain mixture D; Solvent replenishment and pre-filtration: Add the remaining acetate-sodium acetate buffer solution to mixture D (to bring the total weight to 1000 parts), stir for 7 minutes, and then pre-filter through a 0.45 μm cellulose membrane to obtain the pre-filtered solution; Ultrasonic homogenization: Place the pre-filtered liquid in an ultrasonic homogenizer and sonicate at 300W and 33℃ for 15 minutes (3 seconds working, 2 seconds intermittent) to obtain a homogenized liquid. Fine filtration and filling: The homogenized solution is finely filtered through a 0.22μm sterile filter membrane, filled into 15mL light-proof pharmaceutical bottles in a Class 100 clean room, and sealed.

[0055] Features: Chitosan nanoparticles are used as a carrier to enhance the retention of active ingredients by utilizing their mucosal adhesion and biocompatibility; Cnidium monnieri extract and crotonin are introduced to synergistically fight viruses, and Viola yedoensis extract is used to enhance the anti-inflammatory effect; ultrasonic homogenization is used instead of high-pressure homogenization, which is suitable for small-scale production.

[0056] Example 5 (using a compound plant essential oil system) A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 8 parts (10% crotonin content) Tea tree oil: 2 parts (40% eucalyptol content) Dipotassium glycyrrhizate: 4 parts (purity ≥ 98%) Purslane extract: 5 portions (purslane glycoside content 7%) Penetration enhancer: 5 parts (1 part laurocapram + 4 parts glycerin, weight ratio 1:4) Preservative: 0.4 parts (0.2 parts phenoxyethanol + 0.2 parts chlorphenesin) pH adjuster: appropriate amount (0.1 mol / L sodium hydroxide solution, adjust pH to 7.2) Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer: Balance (make up to 1000 parts, pH 7.3) Its preparation method includes the following steps: Preparation of carrier solution: Take 4 parts of dipotassium glycyrrhizate, add 500 parts of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer (pH 7.3), stir at 240 rpm for 16 min in a constant temperature water bath at 34℃ until completely dissolved to obtain the carrier solution; Plant extracts and essential oils were combined: 8 parts of Brucea javanica extract and 5 parts of Portulaca oleracea extract were mixed evenly and added to a carrier solution; 2 parts of tea tree essential oil were dissolved in 10 parts of ethanol and slowly added dropwise to the above solution. The mixture was stirred at 330 rpm for 38 minutes at 34°C to obtain mixture A. Addition of penetration enhancer: Take 1 part laurocapram and 4 parts glycerin, mix them evenly, add them to mixture A, and stir at 290 rpm for 19 min at 34℃ to obtain mixture B; pH adjustment: Slowly add 0.1 mol / L sodium hydroxide solution to mixture B, stir at 190 rpm until the pH value stabilizes at 7.2, to obtain mixture C; Preservation stage: Add phenoxyethanol and chlorphenesin to mixture C, stir at 190 rpm for 9 min to obtain mixture D; Solvent replenishment and pre-filtration: Add the remaining buffer solution to mixture D (to make the total weight 1000 parts), stir for 9 min, and then pre-filter through a 0.45 μm polyvinylidene fluoride filter membrane to obtain the pre-filtered solution; High-pressure homogenization: The pre-filtered liquid is fed into a high-pressure homogenizer and homogenized three times at 34 MPa and 34 °C for 7 minutes each time to obtain a homogenized liquid; Fine filtration and filling: The homogenized solution is finely filtered through a 0.22μm sterile filter membrane, filled into 20mL light-proof pharmaceutical bottles in a Class 100 clean room, and sealed.

[0057] Features: It uses dipotassium glycyrrhizate as a natural carrier, which has both anti-inflammatory and solubilizing effects; it introduces tea tree oil to enhance anti-HPV activity, and forms a synergistic effect of fat-soluble and water-soluble components with Brucea javanica extract; it strengthens the soothing and repairing function of purslane extract, making it suitable for long-term use on sensitive mucous membranes.

[0058] The two embodiments above, by changing the carrier type, the combination of plant ingredients, and some process parameters, provide solutions that adapt to different production conditions and application scenarios while maintaining the core anti-HPV efficacy, further verifying the flexibility and scalability of the technical solution of the present invention.

[0059] Example 6 (using hyaluronic acid-gelatin composite carrier) A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 15 parts (Brucea javanica content 13%) Isatis root extract: 7 parts (5% indirubin content) Hyaluronic acid-gelatin complex: 6 parts (hyaluronic acid to gelatin mass ratio 1:2) Honeysuckle extract: 4 parts (7% chlorogenic acid content) Penetration enhancer: 2 parts (0.5 parts octyldodecyl alcohol + 1.5 parts polyethylene glycol 400) Preservative: 0.3 parts (benzyl alcohol) pH adjuster: appropriate amount (0.1 mol / L phosphoric acid solution, adjust pH to 7.0) Citric acid-sodium citrate buffer: Balance (make up to 1000 parts, pH 6.8) Its preparation method includes the following steps: Carrier solution preparation: 6 parts of hyaluronic acid-gelatin complex were added to 550 parts of buffer solution and stirred at 210 rpm for 25 min at 31°C until completely swollen to obtain the carrier solution; Plant extract compounding: Mix 15 parts of Brucea javanica extract, 7 parts of Isatis indigotica extract, and 4 parts of Lonicera japonica extract, add carrier solution, and stir at 310 rpm for 36 min at 31℃ to obtain mixture A; Penetration enhancer addition: Mix octyl dodecanol with polyethylene glycol 400 and add to mixture A. Stir at 260 rpm for 17 min to obtain mixture B. pH adjustment: Add phosphoric acid solution to adjust the pH to 7.0, stir at 160 rpm to obtain mixture C; Preservative and subsequent processing: Add 0.3 parts benzyl alcohol and stir for 6 min, add buffer to 1000 parts, filter through a 0.45 μm filter membrane, homogenize under high pressure (31 MPa, 31 °C, 2 times), and finally filter and fill through a 0.22 μm filter membrane.

[0060] Features: The hyaluronic acid-gelatin composite carrier has both adhesive and moisturizing properties, Isatis root extract enhances the antiviral spectrum (with significant effects on HPV types 6 / 11), and honeysuckle extract strengthens the broad-spectrum antibacterial effect, making it suitable for HPV patients with concurrent bacterial infections.

[0061] Example 7 (using nanoliposome encapsulation technology) A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 9 parts (Brucea javanica content 12%) Curcuma oil: 3 parts (curcumol content 20%) Nanoliposomes: 10 parts (phospholipid to cholesterol mass ratio 5:1, particle size 100-150nm) Sophora flavescens extract: 5 parts (oxymatrine content 15%) Penetration enhancer: 4 parts (1 part decyl glucoside + 3 parts propylene glycol) Preservative: 0.25 parts (potassium sorbate) pH adjuster: appropriate amount (0.1 mol / L sodium hydroxide solution, adjust pH to 6.7) Tris-HCl buffer: Balance (make up to 1000 samples, pH 7.2) Its preparation method includes the following steps: Preparation of drug-loaded liposomes: 10 parts of nanoliposomes were mixed with 9 parts of Brucea javanica extract and 3 parts of Curcuma zedoaria oil, and the mixture was ultrasonically treated at 35°C for 20 min (power 250W) to achieve encapsulation, resulting in a drug-loaded liposome suspension. Aqueous phase mixing: Dissolve 5 parts of Sophora flavescens extract in 500 parts of Tris-HCl buffer, add drug-loaded liposome suspension, and stir at 300 rpm for 30 min at 30℃. Subsequent processes: Permeation enhancer was added sequentially (stirred at 280 rpm for 18 min), pH was adjusted (stirred at 170 rpm), and preservative was added (stirred at 170 rpm for 8 min). After adding buffer, the mixture was filtered through a 0.45 μm filter membrane, homogenized under high pressure (33 MPa, 33 °C, 3 times), and finally finely filtered and filled.

[0062] Features: Nanoliposome encapsulation enhances the stability of lipid-soluble components; Curcuma zedoaria oil and crotonin synergistically disrupt viral structure; Sophora flavescens extract enhances immunomodulatory function, making it suitable for HPV infection intervention in immunocompromised individuals.

[0063] Example 8 (using a plant-derived composite carrier system) A plant-based compound antibacterial liquid composition for HPV infection, with a total weight of 1000 parts, the weight parts of each component are as follows: Brucea javanica extract: 11 parts (Brucea javanica content 11%) Pomegranate peel extract: 6 parts (ellagic acid content 18%) Pectin-gum arabic complex: 7 parts (1:1 by weight) Forsythia extract: 3 portions (forsythoside content 9%) Penetration enhancer: 3 parts (0.6 parts oleyl alcohol polyoxyethylene ether + 2.4 parts glycerin) Preservative: 0.35 parts (sodium dehydroacetate) pH adjuster: appropriate amount (0.1 mol / L hydrochloric acid solution, adjust pH to 7.3) Potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer: Balance (make up to 1000 parts, pH 7.4) Its preparation method includes the following steps: Carrier solution preparation: 7 parts of pectin-gum arabic complex were added to 580 parts of buffer solution and stirred at 230 rpm for 22 min at 32°C until a colloidal solution was formed; Plant extract compound: Mix 11 parts of Brucea javanica extract, 6 parts of pomegranate peel extract, and 3 parts of Forsythia suspensa extract, add to carrier solution, and stir at 320 rpm for 34 min at 34℃. Subsequent processes: Add penetration enhancer (stir at 270 rpm for 16 min), adjust pH (stir at 180 rpm), add preservative (stir at 180 rpm for 7 min), add buffer solution, filter through a 0.45 μm filter membrane, homogenize under high pressure (32 MPa, 32 °C, twice), and finally fine filter and fill.

[0064] Features: It uses a fully plant-derived carrier (pectin-gum arabic) to improve biocompatibility; ellagic acid from pomegranate peel extract enhances antioxidant and viral inhibition effects; and forsythia extract strengthens anti-inflammatory and swelling-reducing effects, making it suitable for HPV infection care in pregnant and lactating women.

[0065] The above embodiments, through innovative carrier technology, expanded combinations of plant ingredients, and optimized process parameters, provide specialized solutions for scenarios such as co-infection, weakened immunity, and special populations while maintaining the core anti-HPV efficacy, further demonstrating the technical flexibility and wide applicability of the present invention.

[0066] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A plant-based compound antibacterial liquid composition targeting HPV infection, characterized in that, By weight, it comprises the following components: 5-20 parts of Brucea javanica extract, 3-15 parts of Manjacali extract, 2-10 parts of acid-anhydrated β-lactoglobulin, 1-8 parts of Taraxacum mongolicum extract, 1-5 parts of penetration enhancer, 0.1-1 parts of pH adjuster, 0.1-0.5 parts of preservative, with the balance being phosphate buffer.

2. A method for preparing a plant-based compound antibacterial liquid composition targeting HPV infection, characterized in that, Includes the following steps: Carrier solution preparation: Take 2-10 parts of anhydride-modified β-lactoglobulin, add 500-600 parts of phosphate buffer, stir at 30-35℃ and 200-250 rpm for 15-20 min until dissolved to obtain the carrier solution; Plant extract compound: Take 5-20 parts of Brucea javanica extract, 3-15 parts of Manjacali extract, and 1-8 parts of Taraxacum mongolicum extract, add them to the carrier solution, and stir at 300-350 rpm for 30-40 min to obtain mixture A; Adding a penetration enhancer: Add 1-5 parts of a penetration enhancer to mixture A, stir at 250-300 rpm for 15-20 minutes to obtain mixture B; pH adjustment: Add 0.1-1 part of pH adjuster to mixture B, stir at 150-200 rpm to adjust the pH to 6.5-7.5, to obtain mixture C; Preservative: Add 0.1-0.5 parts of preservative to mixture C, stir at 150-200 rpm for 5-10 minutes to obtain mixture D; Solvent replenishment and pre-filtration: Add the remaining phosphate buffer to mixture D to a total weight of 1000 parts, stir, and then filter through a 0.45 μm filter membrane to obtain the pre-filtered solution; High-pressure homogenization: Homogenize the pre-filtered liquid 2-3 times under a pressure of 30-35 MPa, each time for 5-8 minutes, to obtain a homogenized liquid; Fine filtration and filling: The homogenized liquid is filtered through a 0.22μm sterile filter membrane, filled into light-proof pharmaceutical bottles under sterile conditions, and sealed.

3. The antibacterial liquid composition according to claim 1, characterized in that, The extract of Brucea javanica was prepared by reflux extraction with an ethanol-water mixed solvent, the volume ratio of ethanol to water was 7:3, the extraction temperature was 70-80℃, the extraction time was 2-3h, and after purification with macroporous resin, the content of crotonin was ≥10%.

4. The antibacterial liquid composition according to claim 1, characterized in that, The mangacali extract was prepared by supercritical CO2 extraction at an extraction temperature of 35-45℃ and an extraction pressure of 25-35MPa. The entrainer was a 10% (v / v) aqueous ethanol solution, and the mangacali glycoside content in the extract was ≥8%.

5. The antibacterial liquid composition according to claim 1, characterized in that, The preparation method of the anhydride-modified β-lactoglobulin is as follows: β-lactoglobulin is dissolved in an acetate-sodium acetate buffer solution at pH 6.0-6.5, maleic anhydride is added, and the mass ratio of β-lactoglobulin to maleic anhydride is 10:1-15:

1. The reaction is carried out at 30-35℃ for 2-3 hours, and after dialyzing, it is freeze-dried to obtain the product, which has an anhydride degree of 20-30%.

6. The antibacterial liquid composition according to claim 1, characterized in that, The dandelion extract was prepared by water decoction-ethanol precipitation method. Water was added and decocted twice, each time for 1.5-2 hours. The decoctions were combined, concentrated, and then ethanol was added to a volume fraction of 70%. The mixture was allowed to stand and precipitate. The supernatant was collected, concentrated, and dried. The chlorogenic acid content was ≥5%.

7. The antibacterial liquid composition according to claim 1, characterized in that, The penetration enhancer is a compound of azone and propylene glycol in a weight ratio of 1:3 to 1:

5.

8. The antibacterial liquid composition according to claim 1, characterized in that, The pH value of the phosphate buffer solution is 7.2-7.4, and the pH value of the composition is 6.5-7.

5.

9. The antibacterial liquid composition according to claim 1, characterized in that, The pH adjuster is selected from at least one of sodium hydroxide and hydrochloric acid; the preservative is selected from at least one of phenoxyethanol and ethylparaben.

10. The preparation method according to claim 2, characterized in that, The high-pressure homogenization temperature in step 7) is 30-35℃; the light-proof medicine bottle in step 8) is a brown glass bottle, and the filling environment is a Class 100 sterile clean room.

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