Brucea javanica extract-cordycepin HPV attenuated preparation as well as preparation method and application thereof

Abstract

The invention discloses a brucea javanica extract-cordycepin HPV (human papillomavirus) attenuated preparation as well as a preparation method and application thereof, and particularly relates to the technical field of antiviral preparations and gynecological medical treatment. The buccal tablet is prepared from the following components: 5%-15% of a brucea javanica extract, 0.3%-1.0% of cordycepin, 1%-3% of a surfactant, 2%-5% of a penetration enhancer, 0.5%-2% of a thickening agent, a pH regulator and the balance of purified water, the dosage of the pH regulator is a dosage for stabilizing the pH value of the preparation in a range of 4.0-6.5. Through pharmacological synergy of the brucea javanica extract and the cordycepin, a breakthrough of dual effects of direct virus resistance and active immune regulation is realized, the virus load is rapidly reduced, and the immune microenvironment of HPV persistent infection is fundamentally reversed by enhancing the self-cleaning capability of the body, so that the immune effect of the human body is improved. Through cooperation of double strategies of physical particle size optimization and chemical penetration enhancement, it is ensured that high-activity medicine components can efficiently permeate and be enriched in a focus part, and therefore the exertion speed and the action strength of the medicine effect are remarkably improved.

Description

Technical Field

[0001] This invention relates to the fields of antiviral agents and gynecological medical technology. More specifically, this invention relates to a croton extract-cordycepin HPV attenuation agent, its preparation method, and its application. Background Technology

[0002] HPV infection is a common reproductive tract infection in women. Persistent infection with high-risk HPV can induce cervical intraepithelial neoplasia (CIN) and even progress to cervical cancer, seriously threatening women's health. The clearance of HPV infection depends on the body's immunity, especially the defense function of the vaginal local immune barrier. Low immunity is one of the key factors for persistent HPV infection and difficulty in achieving negative clearance. Currently, commonly used clinical treatments for HPV-related diseases include antiviral drugs, immunomodulators, and physical therapy. Therefore, for the treatment of HPV infection and related lesions, how to effectively activate and enhance local mucosal immunity while inhibiting viral replication has become the key to improving the clinical clearance rate, preventing recurrence, and reversing CIN.

[0003] Current clinical treatment options for HPV infection are significantly inadequate. Antiviral drugs such as interferon are prone to drug resistance and have significant side effects; physical therapies such as LEEP are invasive to tissues and have difficulty eliminating latent viruses; and existing preparations based on natural ingredients such as Brucea javanica generally suffer from core problems such as crude extraction processes leading to low retention of active ingredients, excessively large particle size resulting in poor transdermal absorption, and single-formulation formulas that cannot effectively address the patient's low local immunity. This leads to unsatisfactory HPV clearance rates and cervical intraepithelial neoplasia reversal rates in existing preparations. Summary of the Invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a croton extract-cordycepin HPV detoxification preparation, its preparation method and application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a Brucea javanica extract-cordycepin HPV detoxification preparation, characterized in that, by mass percentage, it is composed of the following components: 5%-15% Brucea javanica extract, 0.3%-1.0% cordycepin, 1%-3% surfactant, 2%-5% penetration enhancer, 0.5%-2% thickener, pH adjuster, and the balance being purified water; The amount of pH adjuster used is the amount used to stabilize the pH value of the preparation within the range of 4.0-6.5; The Brucea javanica extract is prepared from Brucea javanica fruit through water extraction, vacuum concentration, and spray drying, wherein the content of crotonin is ≥1.8%, and the retention rate of antiviral active ingredients is ≥92%. The purity of the cordycepin is ≥98%; The formulation has a particle size ≤200nm, a pH value of 4.0-6.5, and contains no added chemical or natural preservatives; The cordycepin is used to target and activate the activity of local macrophages and NK cells in the vagina.

[0006] The core active ingredients are a combination of Brucea javanica extract and cordycepin. The content of crotonin is ≥1.8%, and the retention rate of active ingredients is ≥92%, which can directly inhibit HPV replication. The amount of cordycepin added is 0.3%-1.0%, preferably 0.5%, with a purity ≥98%. It can target and activate local vaginal immune cells, increase macrophage phagocytosis rate, and enhance NK cell activity. By regulating the local immune microenvironment, it synergistically accelerates HPV clearance with crotonin, overcoming the deficiency of traditional preparations that only kill viruses without modulating immunity. Simultaneously, both the extract and cordycepin have certain antibacterial activity, providing a basis for a preservative-free formulation.

[0007] Preferably, the surfactant is poloxamer 188; the penetration enhancer is a mixture of ethanol and propylene glycol in a volume ratio of 1:1 to 2:1; the thickener is carbomer 940; and the pH adjuster is a combination of sodium hydroxide solution and lactic acid.

[0008] The surfactant used is poloxamer 188 to improve the dispersibility of the ingredients; the penetration enhancer is a combination of ethanol and propylene glycol, which synergistically enhances transdermal absorption and also has antibacterial effects; the thickener is carbomer 940 to prolong the retention time of the formulation; the pH adjuster is adjusted to 4.0-6.5 by first using an alkaline solution and then an acidic solution to match the physiological environment of the vagina, inhibit the growth of pathogenic bacteria, and ensure the stability of the preservative-free system.

[0009] Preferably, the cordycepin is prepared by purifying an artificially fermented Cordyceps militaris extract with a purity of ≥99%.

[0010] A method for preparing a *Brucea javanica* extract-cordycepin HPV detoxification agent as described above, characterized by comprising the following steps: S1: Preparation of Brucea javanica extract: Select dried and mature Brucea javanica fruits, pulverize to 40-60 mesh, add purified water at a material-to-liquid ratio of 1:15-1:25, and extract at 90-95℃ 2-3 times, 1.5-2 hours each time, and combine the extracts; concentrate the combined extract under reduced pressure at a vacuum of 0.06-0.08MPa and a temperature of 55-65℃ to a concentrate with a relative density of 1.15-1.25 at 60℃; then spray dry at an inlet air temperature of 160-170℃ and an outlet air temperature of 70-80℃ to obtain Brucea javanica extract powder; S2: Preparation of the mixture: Weigh the Brucea javanica extract powder, cordycepin, and surfactant according to the mass percentages described in claim 1, add purified water equivalent to 20%-30% of the total mass of the preparation, and stir at 30-40°C for 30-45 minutes until the mixture is uniform. S3: Wet milling and homogenization. The mixture obtained in S2 is wet milled, and the grinding temperature is controlled at ≤70℃ until the particle size of the material is ≤2μm. Then the wet-milled material is homogenized under high pressure. The homogenization is carried out 2-3 times under the conditions of ≤60℃ and 120-180bar pressure until the particle size of the material is ≤200nm. S4: Dehydration and thickening: The homogenized material is vacuum evaporated at 50-60℃ and 0.07-0.09MPa to remove moisture, so that the material mass reaches 80%-85% of the total mass of the final formulation; then the penetration enhancer and thickener in the proportions described in claim 1 are added, and the mixture is stirred at 40-50℃ for 60-90 minutes to form a uniform colloid; S5: pH adjustment and dispensing. Add a pH adjuster to the colloid obtained in S4. First, add sodium hydroxide solution to adjust the pH to 7.0-8.0, then add lactic acid or citric acid solution to adjust the pH to 4.0-6.5. Finally, dispense the pH-adjusted colloid into a sterile drug delivery device and seal it for storage.

[0011] Preferably, the material-to-liquid ratio in S1 is 1:20, the extraction is performed 3 times, and the extraction time for each extraction is 2 hours; the inlet air temperature of the spray dryer is 165°C, and the outlet air temperature is 75°C.

[0012] Preferably, in step S3, the high-pressure homogenization pressure is 150 bar, and the homogenization is performed 3 times; in step S2, the amount of cordycepin added is 0.5% of the total mass of the preparation, and the stirring speed is 350 r / min; in step S4, the penetration enhancer is a mixture of ethanol and propylene glycol in a volume ratio of 1.5:1.

[0013] A method for determining the content of Brucea javanica extract and cordycepin in the above-mentioned preparation, using high performance liquid chromatography for simultaneous determination, the specific steps of which are as follows: (1) Chromatographic conditions: The chromatographic column was a C18 column with a size of 4.6 mm × 250 mm and a diameter of 5 μm; the mobile phase was methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 55:45; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelengths were 270 nm for crotonin and 260 nm for cordycepin; the injection volume was 20 μL. (2) Standard curve preparation: Accurately weigh appropriate amounts of crotonin standard and cordycepin standard, dissolve them in methanol and dilute to volume to prepare crotonin standard stock solution with a concentration of 0.1 mg / mL and cordycepin standard stock solution with a concentration of 0.05 mg / mL; accurately pipette 0.1, 0.2, 0.5, 1.0, and 2.0 mL of the above stock solutions into 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to obtain a series of standard working solutions; inject and determine according to the above chromatographic conditions, plot the standard curve with peak area as the ordinate and concentration as the abscissa, and obtain the regression equation; (3) Sample determination: Accurately weigh 10 mg of the preparation sample, place it in a stoppered conical flask, accurately add 25 mL of methanol, weigh it, sonicate for 30 minutes, sonication power 250 W, frequency 40 kHz, cool, weigh it again, replenish the lost weight with methanol, shake well, filter, take the filtrate and inject it for determination under the above chromatographic conditions, record the peak area, and substitute it into the corresponding regression equation to calculate the content of crotonin and cordycepin.

[0014] Preferably, in step (1), the mobile phase is methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 55:45.

[0015] Preferably, the recoveries of crotonin and cordycepin in the method are 98.2%-101.5%, and the relative standard deviations (RSDs) are both no greater than 1.8%. The use of a Cordycepin-HPV attenuated preparation of Brucea javanica extract as described above in the preparation of a drug for treating human papillomavirus (HPV) infection or reversing cervical intraepithelial neoplasia (CIN).

[0016] The technical effects and advantages of this invention are as follows: Through the pharmacological synergy of Brucea javanica extract and cordycepin, a breakthrough in the dual efficacy of direct antiviral and active immune regulation has been achieved. The symptomatic effect of Brucea javanica directly inhibiting viral replication, combined with the fundamental effect of cordycepin targeting and activating the function of local vaginal immune cells such as macrophages and NK cells, not only rapidly reduces viral load, but also fundamentally reverses the immune microenvironment of persistent HPV infection by enhancing the body's own clearance ability. This solves the core defects of traditional preparations that only treat but do not prevent and are prone to recurrence, and achieves a revolution in treatment strategy. By combining nanotechnology with a penetration enhancer system, the bioavailability and targeting of the drug are significantly improved. The combined process of wet milling and high-pressure homogenization enables the active ingredients to reach nanoscale particle size, greatly increasing their contact area with mucosal tissue. At the same time, the optimized compounded ethanol-propylene glycol penetration enhancer system further enhances the synergistic effect, effectively overcoming the cervical mucosal barrier. This dual strategy of physical particle size optimization and chemical penetration enhancement ensures that the highly active drug components can efficiently penetrate and accumulate at the lesion site, thereby significantly improving the speed and intensity of drug efficacy. Through the synergistic antibacterial action of active ingredients, excipients, and pH adjustment, a safe and non-irritating stable system is achieved while ensuring high efficacy. By utilizing the natural antibacterial activity of Brucea javanica extract and cordycepin, combined with the auxiliary antibacterial effect of ethanol-propylene glycol permeation enhancer, and by maintaining a healthy acidic environment in the vagina through precise pH control, a multi-layered and synergistic antibacterial protection is formed. This synergistic design allows the formulation to remain stable during long-term storage without relying on any added chemical or natural preservatives, while perfectly matching the physiological environment of the vagina, fundamentally avoiding the irritation risks caused by preservatives, and achieving a balance between high efficacy and safety. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation method of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Technical solution of the present invention 1. Formulation component design and optimization The core active ingredients are combined for antiviral and immune enhancement: Brucea javanica extract and cordycepin are selected as the core active ingredients. The content of crotonin is ≥1.8%, and the retention rate of active ingredients is ≥92%, which can directly inhibit HPV replication. The cordycepin addition is 0.3%-1.0%, preferably 0.5%, with a purity ≥98%. It can target and activate local vaginal immune cells, increasing macrophage phagocytosis and enhancing NK cell activity. By regulating the local immune microenvironment, it synergistically accelerates HPV clearance with crotonin, overcoming the deficiency of traditional preparations that only kill viruses without modulating immunity. Simultaneously, both the extract and cordycepin have certain antibacterial activity, providing a basis for a preservative-free formulation.

[0020] The excipient system was optimized and designed to be preservative-free: Poloxamer 188 was selected as the surfactant to improve the dispersibility of the ingredients; the penetration enhancer adopted a complex system of ethanol and propylene glycol to synergistically enhance transdermal absorption and also have antibacterial effects; Carbomer 940 was selected as the thickener to prolong the retention time of the formulation; the pH adjuster was adjusted to 4.0-6.5 by first using an alkaline solution and then an acidic solution to match the physiological environment of the vagina, inhibit the growth of pathogenic bacteria, and ensure the stability of the preservative-free system.

[0021] 2. Optimization of preparation method and comparison of parameters Extraction preparation optimization: The material-to-liquid ratio was determined to be 1:20 (g / mL), with 3 extractions and 2 hours each time. The extraction rate of crotonin reached 1.92%, and the retention rate was ≥92%. Low-temperature vacuum concentration and spray drying were combined to reduce component degradation.

[0022] Active ingredient compounding process: Cordycepin is stirred and dissolved together with Brucea javanica extract and surfactant at 30-40℃, with the stirring speed controlled at 350r / min to ensure uniform dispersion and avoid excessive local concentration that may affect safety and efficacy.

[0023] The wet milling and high-pressure homogenization combined process: the optimal parameters are a pressure of 150 bar and three homogenizations, resulting in a formulation particle size of 185 nm, which significantly improves transdermal absorption efficiency. At the same time, the high-pressure environment kills some microorganisms and ensures sterility.

[0024] Other process optimizations: medium-temperature vacuum evaporation reduces water activity, and precise pH adjustment balances activity, safety, and corrosion prevention requirements.

[0025] 3. Methods for determining the content of active ingredients The contents of crocin and cordycepin were simultaneously determined by HPLC. The chromatographic conditions were clearly defined, and dual detection wavelengths were used: 270 nm / 260 nm. This method achieved a recovery rate of 98.2%–101.5% and an RSD ≤ 1.8%, enabling accurate quantification of both components and ensuring batch-to-batch stability of the product. Example 1: Preparation of a Cordycepin-based HPV detoxification agent derived from Brucea javanica extract. 1. Preparation of Brucea javanica extract: Select 1000 grams of dried, mature, and mold-free Brucea javanica fruit and grind it to about 50 mesh using a universal grinder.

[0026] Place the crushed Brucea javanica powder in a multi-functional extraction tank and add 20 liters of purified water at a material-to-liquid ratio of 1:20.

[0027] Turn on heating and stirring, and perform hot reflux extraction at 95℃. Extract for a total of 3 times, with each extraction lasting 2 hours.

[0028] The three extracts were combined and filtered through a 200-mesh filter cloth to obtain a clear extract.

[0029] The filtrate was transferred to a vacuum concentrator and concentrated under reduced pressure at a vacuum of 0.07 MPa and a temperature of 60°C until the relative density of the concentrate was measured to be 1.20.

[0030] The concentrate was spray-dried with the following parameters: inlet air temperature 165℃, outlet air temperature 75℃, and feed pump frequency 20Hz. Approximately 128 grams of brownish-yellow, uniformly textured Brucea javanica extract powder were obtained.

[0031] The content of crotonin was determined to be 1.92% by high performance liquid chromatography (HPLC), and its retention rate of antiviral active ingredients relative to the raw material was calculated to be 93.5%.

[0032] 2. Preparation of the mixture: Based on a final total weight of 1000 grams, accurately weigh the following components: Brucea javanica extract powder: 100g, accounting for 10%; Cordycepin with a purity of ≥99%: 5 grams, accounting for 0.5%; Poloxamer 188: 20 grams, accounting for 2%; Add the above materials to the mixing tank, and add 250 grams of purified water, which is 25% of the total system mass.

[0033] Stir at 350 rpm for 40 minutes in a 35℃ water bath until cordycepin is completely dissolved, the material is evenly mixed, and there are no visible particles.

[0034] 3. Wet milling and homogenization: Transfer the mixture into a vertical ball mill, and add an appropriate amount of purified water to bring the total system volume to approximately 800 mL. Control the temperature during the grinding process to not exceed 65°C, and wet grind until the material particle size is ≤2 micrometers.

[0035] The wet-milled slurry was transferred to a low-temperature high-pressure homogenizer and subjected to high-pressure homogenization and circulation at 55°C and 150 bar for a total of 3 homogenization cycles.

[0036] The particle size distribution of the obtained nanoemulsion was measured by a laser particle size analyzer. The particle size distribution was 180-190 nanometers, and the average particle size was 185 nanometers, which meets the requirement of ≤200 nanometers.

[0037] 4. Remove moisture and thicken: The homogenized nanoemulsion was transferred to a vacuum evaporator and subjected to medium-temperature vacuum evaporation at 55°C and 0.08 MPa to remove some of the moisture until the total mass of the material was reduced to about 820 grams, which is about 82% of the total mass of the final formulation.

[0038] Add the following to the concentrated material: Ethanol-propylene glycol compound penetration enhancer with a volume ratio of 1.5:1: 30 g, accounting for 3%; Carbomer 940: 10 grams, accounting for 1%; Stir at 45°C at a medium speed for 75 minutes until the carbomer is completely swollen, forming a uniform, translucent gel-like colloid.

[0039] 5. pH adjustment and dispensing: Slowly add 0.1 mol / L sodium hydroxide solution to the colloidal system while stirring, and adjust the pH of the system to 7.5.

[0040] Then, slowly add 0.1 mol / L citric acid or lactic acid solution dropwise to precisely adjust the pH value to 4.8, which is within the physiologically compatible pH range of 4.0-6.5 for the vagina. Continue stirring for 20 minutes to ensure that the pH value is uniform and stable. The deviation should be ≤ ±0.1 as measured by a precision pH meter.

[0041] The pH-adjusted formulation was filtered and sterilized in a Class 100 clean environment through a 0.22-micron polyethersulfone filter membrane.

[0042] Using aseptic filling equipment, each 5-gram vial is filled into a pre-sterilized disposable vaginal applicator, sealed, and then sterilized by cobalt-60 irradiation at a dose of 25 kGy to obtain the finished product of Brucea javanica extract-cordycepin HPV attenuated preparation.

[0043] Example 2: Content determination method and stability verification This example illustrates the method for determining the content of active ingredients in a formulation and the stability of the product.

[0044] Content determination: Chromatographic conditions: An Agilent 1260 high-performance liquid chromatography system was used. Column: Diamonsil C18, 4.6 mm × 250 mm, 5 μm; mobile phase: methanol-0.1% phosphoric acid aqueous solution, 55:45; flow rate: 1.0 mL / min; column temperature: 30 ℃; detection wavelength: cordycepin for 0-12 minutes, 260 nm; crotonin for 12-25 minutes, 270 nm; injection volume: 20 μL.

[0045] Standard curve preparation: Accurately weigh 10.0 mg of crotonin standard and 5.0 mg of cordycepin standard, dissolve them in methanol and dilute to 100 mL volumetric flasks to obtain standard stock solutions with concentrations of 0.1 mg / mL and 0.05 mg / mL, respectively. Accurately pipette 0.1, 0.2, 0.5, 1.0, and 2.0 mL of the stock solution into 10 mL volumetric flasks, dilute to volume with methanol, and prepare a series of standard working solutions. Inject the samples under the above chromatographic conditions, and perform linear regression on peak area (Y) against concentration (X, μg / mL) to obtain the regression equation. Crotonin showed good linearity in the range of 1-20 μg / mL, r 2 =0.9998, cordycepin showed good linearity in the range of 0.5-10 μg / mL, r 2 =0.9996.

[0046] Sample determination: Accurately weigh 10.0 mg of the preparation obtained in Example 1, place it in a 25 mL volumetric flask, add approximately 20 mL of methanol, sonicate (power 250 W, frequency 40 kHz) for 30 minutes, cool, dilute to the mark with methanol, shake well, filter through a 0.45 μm microporous membrane, and inject the filtrate for analysis; calculate the content using the external standard method, and determine that the content of crotonin in this product is 1.89% and the content of cordycepin is 0.49%. The average recovery rate of crotonin using this method is 99.3%, RSD=1.1%, and the average recovery rate of cordycepin is 100.5%, RSD=0.9%, indicating high precision and accurate results.

[0047] Stability verification: The three batches of finished formulations prepared in Example 1 were placed in a drug stability test chamber and subjected to long-term sample retention and observation under the conditions of temperature 25±2℃ and relative humidity 60%±5%.

[0048] Samples were taken at the end of 0, 1, 3, 6, 12, and 18 months, and all items were tested according to the quality standards.

[0049] Results: At the end of 18 months, no stratification, mold, or rancidity was observed in any of the three batches of samples; the pH value remained stable at 4.7-4.9; the content of active ingredients was as follows: the average content of crotonin was 97.5% of the initial content, and the average content of cordycepin was 97.8% of the initial content, with degradation rates of <3% for both; the particle size of the formulation remained at 185-198 nanometers. The results indicate that the product maintains stable quality after 18 months of storage at room temperature without the addition of any chemical or natural preservatives, meeting the requirements for clinical application.

[0050] Example 3: Verification of Clinical Application Efficacy This embodiment is used to verify the clinical efficacy and safety of the formulation of the present invention.

[0051] Experimental design: A randomized, controlled, single-blind clinical trial design was adopted.

[0052] Study subjects: 120 female patients aged 20-50 years who were diagnosed by PCR with persistent infection of high-risk HPV16 / 18 for ≥6 months and with cervical intraepithelial neoplasia, CINI-II were recruited. Patients were randomly divided into an experimental group (n=60) and a control group (n=60). There were no significant differences between the two groups in baseline data such as age, duration of infection, and CIN grade (P>0.05).

[0053] Dosage regimen: Experimental group: The preparation of this invention contains 0.5% cordycepin, and is administered vaginally once every two days, one vial each time, one vial is 5g, and the patient lies flat for 30 minutes after administration.

[0054] Control group: A single extract of Brucea javanica without cordycepin was used, with the other components and usage being the same as the experimental group.

[0055] Treatment course: Continuous medication for 3 months, followed by a 3-month follow-up period after discontinuation. Discontinue medication during menstruation.

[0056] Evaluation indicators: HPV seroconversion rate: HPV-DNA was detected by PCR before medication and 3 months after medication.

[0057] CIN reversal rate: CIN grade is assessed by colposcopy and pathological biopsy before and 6 months after medication. Reversal is considered when the pathological results return to normal or drop below CIN I grade.

[0058] Immune indicators: Cervical exfoliated cells were collected before and 3 months after medication. Changes in NK cell activity were detected by flow cytometry, and changes in macrophage phagocytosis rate were detected by phagocytic fluorescent microsphere method.

[0059] Safety: Record all adverse events during medication use.

[0060] result: Evaluation indicators Experimental group (the formulation of this invention) Control group (single preparation of Brucea javanica) p-value 3-month HPV seroconversion rate 87.5%(52 / 60) 61.7%(37 / 60) <0.01 6-month CIN reversal rate 80.0%(48 / 60) 53.3%(32 / 60) <0.01 NK cell activity enhancement rate An average increase of 34.2% An average increase of 7.8% <0.01 Macrophage phagocytic rate increase An average increase of 42.5% An average increase of 11.3% <0.01 Adverse reaction incidence 3.3% (2 / 60, mild and transient burning sensation) 5.0% (3 / 60, mild itching or burning sensation) >0.05 Conclusion: Clinical trial results show that, compared with single-ingredient *Brucea javanica* preparations without cordycepin, the *Brucea javanica* extract-cordycepin compound preparation provided by this invention can significantly improve the HPV seroconversion rate and CIN reversal rate, and effectively activate local vaginal cellular immune function, NK cells and macrophages, with good safety and no serious adverse reactions observed. This fully verifies the key role of cordycepin in synergistic antiviral activity and enhancing local immunity, as well as the synergistic advantage of the dual antiviral and immunomodulatory effects of the preparation of this invention. In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A *Brucea javanica* extract-cordycepin HPV detoxification preparation, characterized in that: By weight percentage, it consists of the following components: 5%-15% Brucea javanica extract, 0.3%-1.0% cordycepin, 1%-3% surfactant, 2%-5% penetration enhancer, 0.5%-2% thickener, pH adjuster, and the balance purified water; The amount of pH adjuster used is the amount used to stabilize the pH value of the preparation within the range of 4.0-6.5; The Brucea javanica extract is prepared by water extraction, vacuum concentration, and spray drying of Brucea javanica fruit, wherein the content of crotonin is ≥1.8%; The purity of the cordycepin is ≥98%; The formulation has a particle size ≤200nm, a pH value of 4.0-6.5, and contains no added chemical or natural preservatives.

2. The *Brucea javanica* extract-cordycepin HPV detoxification preparation according to claim 1, characterized in that: The surfactant is poloxamer 188; the penetration enhancer is a mixture of ethanol and propylene glycol in a volume ratio of 1:1 to 2:1; the thickener is carbomer 940; and the pH adjuster is a combination of sodium hydroxide solution and lactic acid.

3. The HPV detoxification preparation of Brucea javanica extract-cordycepin according to claim 1, characterized in that: The cordycepin is prepared by purifying an artificially fermented and cultured Cordyceps militaris extract, with a purity of ≥99%.

4. A method for preparing the *Brucea javanica* extract-cordycepin HPV detoxification agent as described in claim 1, characterized in that: Includes the following steps: S1: Preparation of Brucea javanica extract: Select dried and mature Brucea javanica fruits, pulverize to 40-60 mesh, add purified water at a material-to-liquid ratio of 1:15-1:25, and extract at 90-95℃ 2-3 times, 1.5-2 hours each time, and combine the extracts; concentrate the combined extract under reduced pressure at a vacuum of 0.06-0.08MPa and a temperature of 55-65℃ to a concentrate with a relative density of 1.15-1.25 at 60℃; then spray dry at an inlet air temperature of 160-170℃ and an outlet air temperature of 70-80℃ to obtain Brucea javanica extract powder; S2: Preparation of the mixture: Weigh the Brucea javanica extract powder, cordycepin, and surfactant according to the mass percentages described in claim 1, add purified water equivalent to 20%-30% of the total mass of the preparation, and stir at 30-40°C for 30-45 minutes until the mixture is uniform. S3: Wet milling and homogenization. The mixture obtained in S2 is wet milled, and the grinding temperature is controlled at ≤70℃ until the particle size of the material is ≤2μm. Then the wet-milled material is homogenized under high pressure. The homogenization is carried out 2-3 times under the conditions of ≤60℃ and 120-180bar pressure until the particle size of the material is ≤200nm. S4: Dehydration and thickening: The homogenized material is vacuum evaporated at 50-60℃ and 0.07-0.09MPa to remove moisture, so that the material mass reaches 80%-85% of the total mass of the final formulation; then the penetration enhancer and thickener in the proportions described in claim 1 are added, and the mixture is stirred at 40-50℃ for 60-90 minutes to form a uniform colloid; S5: pH adjustment and dispensing. Add a pH adjuster to the colloid obtained in S4. First, add sodium hydroxide solution to adjust the pH to 7.0-8.0, then add lactic acid or citric acid solution to adjust the pH to 4.0-6.

5. Finally, dispense the pH-adjusted colloid into a sterile drug delivery device and seal it for storage.

5. The preparation method of the Brucea javanica extract-cordycepin HPV detoxification agent according to claim 4, characterized in that: The material-to-liquid ratio in S1 is 1:20, the extraction is performed 3 times, and the extraction time for each extraction is 2 hours; the inlet air temperature of the spray dryer is 165℃, and the outlet air temperature is 75℃.

6. The preparation method of the Brucea javanica extract-cordycepin HPV detoxification agent according to claim 4, characterized in that: In S3, the high-pressure homogenization pressure is 150 bar, and the homogenization is performed 3 times; in S2, the amount of cordycepin added is 0.5% of the total mass of the preparation, and the stirring speed is 350 r / min; in S4, the penetration enhancer is a mixture of ethanol and propylene glycol with a volume ratio of 1.5:

1.

7. A method for determining the content of Brucea javanica extract and cordycepin in the preparation as described in claim 1, characterized in that: Simultaneous determination using high-performance liquid chromatography (HPLC) is performed as follows: (1) Chromatographic conditions: The chromatographic column was a C18 column with a size of 4.6 mm × 250 mm and a diameter of 5 μm; the mobile phase was methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 55:45; the flow rate was 1.0 mL / min; the column temperature was 30 °C; the detection wavelengths were 270 nm for crotonin and 260 nm for cordycepin; the injection volume was 20 μL. (2) Standard curve preparation: Accurately weigh appropriate amounts of crotonin standard and cordycepin standard, dissolve them in methanol and dilute to volume to prepare crotonin standard stock solution with a concentration of 0.1 mg / mL and cordycepin standard stock solution with a concentration of 0.05 mg / mL; accurately pipette 0.1, 0.2, 0.5, 1.0, and 2.0 mL of the above stock solutions into 10 mL volumetric flasks, dilute to the mark with methanol, and shake well to obtain a series of standard working solutions; inject and determine according to the above chromatographic conditions, plot the standard curve with peak area as the ordinate and concentration as the abscissa, and obtain the regression equation; (3) Sample determination: Accurately weigh 10 mg of the preparation sample, place it in a stoppered conical flask, accurately add 25 mL of methanol, weigh it, sonicate for 30 minutes, sonication power 250 W, frequency 40 kHz, cool, weigh it again, replenish the lost weight with methanol, shake well, filter, take the filtrate and inject it for determination under the above chromatographic conditions, record the peak area, and substitute it into the corresponding regression equation to calculate the content of crotonin and cordycepin.

8. The content determination method according to claim 7, characterized in that: In step (1), the mobile phase is methanol-0.1% phosphoric acid aqueous solution with a volume ratio of 55:

45.

9. The content determination method according to claim 1, characterized in that: The recovery rates of crotonin and cordycepin in the method were 98.2%-101.5%, and the relative standard deviations (RSDs) were all no greater than 1.8%.

10. The use of the Brucea javanica extract-cordycepin HPV attenuated preparation as described in claim 1 in the preparation of a medicament for treating human papillomavirus infection or reversing cervical intraepithelial neoplasia.

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