Functional gynecological dressing for preventing or treating HPV infection

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a pharmaceutical composition for preventing or treating HPV infection, which comprises modified β-lactoglobulin and a fish skin protein short peptide, and the weight ratio of the modified β-lactoglobulin and the fish skin protein short peptide is 1 to 10: 10 to 1. The present invention also provides a functional gynecological dressing comprising the pharmaceutical composition. The present invention further provides the use of the pharmaceutical composition or the functional gynecological dressing in the preparation of a drug for preventing or treating HPV infection, and the drug also has an antibacterial effect. The present invention unexpectedly found that the anti-HPV activity of the modified β-lactoglobulin can be significantly enhanced by combining a specific fish skin protein short peptide (HPLGL) with modified β-lactoglobulin in a specific mass ratio, and it is also found that the pharmaceutical composition obtained after the combination has an antibacterial effect, achieving an unexpected technical effect.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a pharmaceutical composition for preventing or treating HPV infection and use of the pharmaceutical composition in preparing a functional gynecological dressing. Background Art

[0002] Human papillomavirus (HPV) belongs to the papillomaviridae family and is a type of epithelial non-enveloped double-stranded DNA virus. So far, HPV isolated from human skin and mucous membrane tissues can be divided into more than 100 types based on different DNA sequences. It can also be divided into "low-risk" and "high-risk" types based on its carcinogenicity. The former low-risk type with a high prevalence is represented by HPV6, HPV11, etc., which mainly cause benign genital warts and low-grade cervical epithelial necrosis; the latter high-risk type with a high prevalence is represented by HPV16, HPV18, etc., and its infection is the main cause of tissue malignancies, especially cervical cancer.

[0003] At present, there is a lack of drugs that can effectively prevent and control HPV virus infection in the world. The most effective ones are preventive vaccines. It has been clinically proven that HPV vaccines can almost 100% prevent infection of some HPV virus strains, but they cannot prevent infection of all types of virus strains, and the price is relatively high. Early treatment of HPV virus infection is very important, but in the absence of specific drugs, immunomodulatory drug therapy and surgical therapy are more commonly used for early treatment of infection. Immunomodulatory drugs are mainly recombinant human interferon α2b, a commonly used antiviral drug in clinical practice, which can inhibit HPV-DNA transcription, thereby blocking the proliferation and division pathway of HPV, so that HPV can be effectively eliminated, but headache, muscle pain, fever, bone marrow suppression and other adverse reactions may occur after medication. The surgery is a loop electrosurgical excision procedure (LEEP). LEEP treatment is performed according to the range of cervical lesions in patients, and the cervical lesion tissue and the entire transformation zone of the patient are removed to the maximum extent. After surgery, the wound surface is electrocoagulated to stop bleeding, but the damage is large, the incidence of postoperative complications is high, and the treatment effect on the long-term recurrence rate of high-risk HPV infection is not obvious.

[0004] Therefore, scientists at home and abroad have spent a lot of money and energy on developing new drugs that have preventive and control effects on multiple HPV strains, but there has been no breakthrough progress. The inventors have previously developed a biological preparation containing modified β-lactoglobulin for preventing and treating HPV virus infection (see CN113171445A). The present invention is a technical upgrade of the previous product. After screening a large number of short peptides derived from fish skin proteins, the inventors unexpectedly found that the combination of a specific fish skin protein short peptide and modified β-lactoglobulin can significantly enhance the anti-HPV activity of the modified β-lactoglobulin, and also found that the composition obtained after the combination has an antibacterial effect.

[0005] On this basis, the inventors have developed a new functional gynecological dressing for preventing or treating HPV infection. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide a safe, effective, simple to prepare, low-cost pharmaceutical composition for preventing or treating HPV infection and its use in preparing functional gynecological dressings.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides a pharmaceutical composition for preventing or treating HPV infection, the pharmaceutical composition comprising modified β-lactoglobulin and a fish skin protein short peptide, wherein the weight ratio of the modified β-lactoglobulin to the fish skin protein short peptide is 1-10:10-1.

[0009] As an optional mode, in the above-mentioned pharmaceutical composition, the weight ratio of the modified β-lactoglobulin to the fish skin protein short peptide is 1-5:5-1.

[0010] As an optional mode, in the above pharmaceutical composition, the weight ratio of the modified β-lactoglobulin to the fish skin protein short peptide is 4:1 or 3:2.

[0011] As an optional manner, in the above pharmaceutical composition, the modified β-lactoglobulin is β-lactoglobulin modified with an active acid anhydride, and the modified β-lactoglobulin can be prepared by the method disclosed in CN113171445A.

[0012] As an optional mode, in the above pharmaceutical composition, the β-lactoglobulin is commercially available, or the β-lactoglobulin can be separated and purified from milk.

[0013] As an optional mode, in the above-mentioned pharmaceutical composition, the modified β-lactoglobulin is β-lactoglobulin modified with active acid anhydride, and the modification method comprises the following steps: adding a saturated solution of active acid anhydride to an aqueous solution of β-lactoglobulin in batches, reacting the saturated solution of active acid anhydride after each addition, and then adjusting the pH to 5-9; dialyzing the anhydride-modified β-lactoglobulin solution with phosphate buffer for 3-5 times to obtain modified β-lactoglobulin.

[0014] As an optional manner, in the above-mentioned pharmaceutical composition, for the preparation method of the modified β-lactoglobulin, the active acid anhydride is 3-hydroxy-phthalic anhydride, the saturated solution of the active acid anhydride is a saturated solution of 3-hydroxy-phthalic anhydride in dimethyl sulfoxide, and the pH adjuster is NaOH, Ca(OH)2, KOH, Na3PO4, ammonium hydroxide, triethanolamine or ethylenediamine.

[0015] As an optional manner, in the above pharmaceutical composition, according to the preparation method of the modified β-lactoglobulin, a saturated solution of 3-hydroxy-phthalic anhydride in dimethyl sulfoxide is added to the β-lactoglobulin aqueous solution in 3 to 8 batches.

[0016] As an optional mode, in the above pharmaceutical composition, for the preparation method of the modified β-lactoglobulin, the anhydride β-lactoglobulin solution is dialyzed 3 times with phosphate buffer, the first dialysis is 2 hours, the second dialysis is 3 hours, and the third dialysis is 12 hours.

[0017] As an optional manner, in the above-mentioned pharmaceutical composition, the amino acid sequence of the fish skin protein short peptide is HPLGL (His-Pro-Leu-Gly-Leu).

[0018] As an optional manner, in the above-mentioned pharmaceutical composition, the fish skin protein short peptide is a tilapia skin enzymatic hydrolysate or is prepared by a solid phase synthesis method.

[0019] In a second aspect, the present invention provides a functional gynecological dressing, comprising the pharmaceutical composition described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0020] As an optional mode, in the above-mentioned functional gynecological dressing, the functional gynecological dressing is a gel preparation, a cream, a spray, a scrub, a toilet wash, etc.

[0021] Preferably, the functional gynecological dressing can be prepared by conventional methods in the field of pharmaceutical preparations.

[0022] As an optional manner, in the above-mentioned functional gynecological dressing, the functional gynecological dressing contains the following components in mass percentage: 0.01% to 0.10% modified β-lactoglobulin, 0.01% to 0.10% fish skin protein short peptide, 3% to 10% glycerol, 3% to 10% anhydrous ethanol, and 0.1% to 1% carbomer.

[0023] In a third aspect, the present invention provides use of the pharmaceutical composition described in the first aspect or the functional gynecological dressing described in the second aspect in the preparation of a medicament for preventing or treating HPV infection.

[0024] As an optional mode, in the above-mentioned use, the drug is used to prevent or treat cervical lesions and genital warts caused by HPV infection and recurrence of the above-mentioned diseases after treatment, and the drug is used to prevent cervical cancer.

[0025] As an option, in the above use, the drug also has an antibacterial effect.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention is a technical upgrade of previous products. After screening a large number of short peptides derived from fish skin proteins, the inventors unexpectedly found that the anti-HPV activity of modified β-lactoglobulin can be significantly enhanced by combining a specific fish skin protein short peptide (HPLGL) with modified β-lactoglobulin in a specific mass ratio. It is also found that the pharmaceutical composition obtained after the combination has an antibacterial effect, achieving unexpected technical effects.

[0028] (2) The inventors have a relatively mature technology for preparing modified β-lactoglobulin, and can prepare high-purity fish skin protein short peptide (HPLGL) using solid phase synthesis technology. Therefore, the preparation method of the raw materials required for preparing the product of the present invention is simple and low in cost.

[0029] (3) The experimental results show that the drug of the present invention can be effectively used to prevent or treat cervical lesions and genital warts caused by HPV infection and the recurrence of the above diseases after treatment. The drug of the present invention can effectively prevent the occurrence of cervical cancer. In addition, the drug of the present invention also has an antibacterial effect. DETAILED DESCRIPTION

[0030] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0031] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0032] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0033] Unless otherwise specified, all percentages and parts in the present invention are percentages and parts by weight.

[0034] Preparation Example:

[0035] 1. Preparation method of modified β-lactoglobulin (see Example 5 in CN113171445A):

[0036] (1) Weigh 250 mg of β-lactoglobulin and dissolve it in 12.5 mL of water to obtain a β-lactoglobulin aqueous solution, wherein the final protein concentration in the β-lactoglobulin aqueous solution is 20 mg / mL;

[0037] (2) dissolving 3-hydroxy-phthalic anhydride in a dimethyl sulfoxide (DMSO) solution to obtain a saturated solution of 3-hydroxy-phthalic anhydride with a saturated concentration of 1.19 M;

[0038] (3) adding a saturated solution of 3-hydroxyphthalic anhydride to the β-lactoglobulin aqueous solution in six portions: adding 318 μL of a saturated solution of 3-hydroxyphthalic anhydride to the β-lactoglobulin aqueous solution for the first time, slowly adding the solution dropwise, reacting for 12 minutes, and then adjusting the pH value to 7.0 with a saturated calcium hydroxide aqueous solution; then adding 318 μL of a saturated solution of 3-hydroxyphthalic anhydride to the β-lactoglobulin aqueous solution for the second time, adjusting the pH value to 6.0, reacting for 12 minutes, and then adjusting the pH value to 7.0 with a saturated calcium hydroxide aqueous solution; adding 318 μL of a saturated solution of 3-hydroxyphthalic anhydride to the β-lactoglobulin aqueous solution for the third time, adjusting the pH value to 6.0, reacting for 12 minutes, and then adjusting the pH value to 7.0 with a saturated calcium hydroxide aqueous solution. The pH value is adjusted to 7.0 with a saturated calcium hydroxide aqueous solution; 318 μL of a saturated 3-hydroxyphthalic anhydride solution is added to the β-lactoglobulin aqueous solution for the fourth time to adjust the pH value to 6.0, then react for 12 minutes, and then adjust the pH value to 7.0 with a saturated calcium hydroxide aqueous solution; 318 μL of a saturated 3-hydroxyphthalic anhydride solution is added to the β-lactoglobulin aqueous solution for the fifth time to adjust the pH value to 6.0, then react for 12 minutes, and then adjust the pH value to 7.0 with a saturated calcium hydroxide aqueous solution; 318 μL of a saturated 3-hydroxyphthalic anhydride solution is added to the β-lactoglobulin aqueous solution for the sixth time to adjust the pH value to 6.0, then react for 12 minutes, and then adjust the pH value to 7.0 with a saturated calcium hydroxide aqueous solution;

[0039] (4) The protein solution after the six reactions with the saturated solution of 3-hydroxy-phthalic anhydride is placed in a dialysis bag and dialyzed in a phosphate buffer solution. The first rotation is for 2 hours, and the dialysate is changed. The second rotation is for 3 hours, and the dialysate is changed. The third dialysis is performed overnight for 12 hours. The protein solution after the three dialysis is the modified β-lactoglobulin, which is refrigerated for later use.

[0040] 2. Preparation method of fish skin protein short peptide (HPLGL):

[0041] 2.1 Preparation of tilapia skin protein peptide:

[0042] Take an appropriate amount of frozen tilapia skin, add water to thaw in a ratio of 1:4 (w / v), then add 5% NaOH to soak and remove impurities, rinse it with clean water for several times until the pH of the washing water is neutral, add 0.05% alkaline protease and 0.05% papain, enzymolyze for 5 hours, then inactivate the enzyme in a boiling water bath for 15 minutes, take the enzymolysis solution and pass it through a 200-mesh silk cloth to remove impurities, filter to remove suspended impurities, then centrifuge at 7500rpm and 4°C for 10 minutes, take the supernatant, and freeze-dry to obtain the tilapia skin enzymolysis product.

[0043] 2.2 Screening and identification of fish skin protein short peptide sequences

[0044] The tilapia skin enzymatic hydrolysate prepared in step 2.1 above was desalted and then subjected to chromatographic separation and mass spectrometry detection.

[0045] The specific conditions are as follows: LC-MS / MS analysis was performed using a Thermo Fisher Scientific dual ternary high performance liquid chromatography (DGLC) tandem quadrupole orbital trap high resolution mass spectrometer (Q-Exactive). The liquid phase was separated by a reverse phase system, with 0.1% formic acid water as the elution phase A and acetonitrile as the elution phase B. The chromatographic column model was: HSS T31.8μm, C18, 100×2.1mm, Waters Acquity, and the elution program was as follows: 0-5.5min: 5%-95%B; 9.0-9.5min: 95%-5%B; 13.0min: 5%B, the flow rate was 0.3mL / min, the column temperature was 35℃, the injection volume was 2μL, the mass spectrometry scanning mode was positive ion, the primary mass spectrometry scanning mass range was 150-1500m / z, the resolution was 35,000, the maximum injection time was 100ms, the MS2 scanning resolution was 17,500, the maximum injection time was 50ms, the collision energy was 35eV, the capillary spray voltage was 3.0kV, the capillary temperature was 320℃, the auxiliary heating temperature was 350℃, and the S-lens was Rf was set to 60. Ten fragmentation spectra (MS2 scans) were collected after each full scan.

[0046] Database search: The mass spectrometry test raw file (Raw File) was searched against the corresponding database using the software MaxQuant1.5.5.1, and finally the protein identification and quantitative analysis results were obtained.

[0047] Mass spectrometry was used to identify the peptide sequences of tilapia skin enzymatic hydrolysates, and peptide sequences with relatively high relative content and high solubility, non-toxicity and good stability predicted by database search were selected for further study.

[0048] 2.3 Preparation of HPLGL (His-Pro-Leu-Gly-Leu) by solid phase synthesis

[0049] The resin was swollen, washed, and dried to remove the Fmoc protecting group. The amino acids that make up the peptide were weighed and reacted in a shaker at 30°C for 1 hour for condensation reaction. The removal-protection-condensation process was repeated until all amino acids were connected. The target peptide was separated from impurities by high performance liquid chromatography (HPLC) to obtain a pure peptide (>99%), and the target peptide was lyophilized into powder.

[0050] 3. Preparation of Example 1

[0051] A functional gynecological dressing comprises the following components in percentage by mass: 0.04% modified beta-lactoglobulin, 0.01% fish skin protein short peptide HPLGL, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer.

[0052] The preparation method of the functional gynecological dressing comprises the following steps: taking the preparation of 1 kg of the dressing as an example, first weighing 0.4 g of modified β-lactoglobulin, 0.1 g of fish skin protein short peptide HPLGL, 30 g of glycerol, 50 g of anhydrous ethanol, and 3 g of carbomer; mixing the modified β-lactoglobulin, carbomer, and an appropriate amount of water to obtain A; then mixing glycerol and anhydrous ethanol to obtain B; mixing A and B, adding water to 1 kg, and stirring evenly to obtain a dressing.

[0053] 4. Preparation of Example 2

[0054] A functional gynecological dressing comprises the following components in percentage by mass: 0.03% modified beta-lactoglobulin, 0.02% fish skin protein short peptide HPLGL, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer.

[0055] The preparation method of the functional gynecological dressing comprises the following steps: taking the preparation of 1 kg of the dressing as an example, first weighing 0.3 g of modified β-lactoglobulin, 0.2 g of fish skin protein short peptide HPLGL, 30 g of glycerol, 50 g of anhydrous ethanol, and 3 g of carbomer; mixing the modified β-lactoglobulin, carbomer, and an appropriate amount of water to obtain A; then mixing glycerol and anhydrous ethanol to obtain B; mixing A and B, adding water to 1 kg, and stirring evenly to obtain a dressing.

[0056] 5. Preparation of Comparative Example 1

[0057] A gynecological dressing comprises the following components in percentage by mass: 0.04% modified beta-lactoglobulin, 0.01% fish skin protein short peptide HKLGL, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer.

[0058] The preparation method of the functional gynecological dressing comprises the following steps: taking the preparation of 1 kg of the dressing as an example, first weighing 0.4 g of modified β-lactoglobulin, 0.1 g of fish skin protein short peptide HKLGL, 30 g of glycerol, 50 g of anhydrous ethanol, and 3 g of carbomer; mixing the modified β-lactoglobulin, carbomer, and an appropriate amount of water to obtain A; then mixing glycerol and anhydrous ethanol to obtain B; mixing A and B, adding water to 1 kg, and stirring evenly to obtain a dressing.

[0059] 6. Preparation of Comparative Example 2

[0060] A gynecological dressing comprises the following components in percentage by mass: 0.05% modified beta-lactoglobulin, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer.

[0061] The preparation method of the functional gynecological dressing comprises the following steps: taking the preparation of 1 kg of the dressing as an example, first weighing 0.5 g of modified β-lactoglobulin, 30 g of glycerol, 50 g of anhydrous ethanol, and 3 g of carbomer; mixing the modified β-lactoglobulin, carbomer, and an appropriate amount of water to obtain A; then mixing glycerol and anhydrous ethanol to obtain B; mixing A and B, adding water to 1 kg, and stirring evenly to obtain a dressing.

[0062] 7. Preparation of Comparative Example 3

[0063] A gynecological dressing comprises the following components in percentage by mass: 0.05% fish skin protein short peptide HPLGL, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer.

[0064] The preparation method of the functional gynecological dressing comprises the following steps: taking the preparation of 1 kg of the dressing as an example, first weighing 0.5 g of fish skin protein short peptide HPLGL, 30 g of glycerol, 50 g of anhydrous ethanol, and 3 g of carbomer; mixing the modified β-lactoglobulin, carbomer, and an appropriate amount of water to obtain A; then mixing glycerol and anhydrous ethanol to obtain B; mixing A and B, adding water to 1 kg, and stirring evenly to obtain a dressing.

[0065] Effect embodiment:

[0066] Effect Example 1: Cytotoxicity Study of the Functional Gynecological Dressing of the Present Invention

[0067] The main experimental methods refer to Example 7 in CN113171445A.

[0068] 1. Main experimental materials

[0069] The samples prepared in the above-mentioned Examples 1-2 and Comparative Examples 1-3 were used for experiments.

[0070] Extraction medium: MEM culture medium containing 10% fetal bovine serum.

[0071] Preparation of test samples: Take Examples 1-2 and Comparative Examples 1-3 respectively, extract at a ratio of 0.2 g: 1 mL at 37°C for 24 h; dilute the obtained extract 8 times to obtain a 12.5% ​​test solution; and soak the test solution with filter paper with a diameter of 5 mm for testing.

[0072] Blank control solution: The same batch of extraction medium was placed at 37℃ for 24h as blank control solution.

[0073] Negative control: Take high-density polyethylene, wash with ultrapure water, dry, and irradiate with ultraviolet light overnight; add the same batch of extraction medium at a ratio of 0.2g / mL and extract at 37℃ for 24h; soak the test solution with filter paper with a diameter of 5mm for testing.

[0074] Positive control: add 0.4 mL of phenol to 7.6 mL of cell culture medium to obtain 5% phenol; soak the test solution with filter paper with a diameter of 5 mm for the test.

[0075] Cell line: Mouse fibroblasts (L-929), provided by the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0076] 2. Experimental Methods

[0077] (1) After culturing for 48 h to 72 h, the vigorously growing cells were digested with digestion solution and added to the cell culture medium. After pipetting and mixing, the cell suspension was prepared to a density of 2.5 × 10 5 / mL, inoculated into 90mm diameter culture dishes, 10mL per dish, 4 dishes in total; cultured in a CO2 incubator for 24h (37℃, 5% CO2); observed cell morphology under a microscope;

[0078] (2) Discard the original culture medium and add 10 mL of freshly prepared 1× agar growth medium; after the agar medium solidifies at room temperature (about 30 minutes), add 10 mL of neutral red vital dye and store in the dark for 30 minutes; remove excess neutral red solution; add one extraction medium, one negative control, one positive control, and one test sample to each dish, and add one extraction medium, one negative control, one positive control, and one test sample to each dish for each test sample (Example 1-2 and Comparative Example 1-3); keep a distance of more than 20 mm between each sample; place the dish in a CO2 incubator and continue to culture for 24 hours;

[0079] (3) After the incubation, the discolored areas around the cell test materials and the control materials were observed under a microscope. The results of each test material were determined according to the grading criteria for cytotoxicity reactions in the agar diffusion test in Table 1. The final reaction result was the median of the reactions of the four parallel samples.

[0080] Table 1: Grading criteria for cytotoxicity in agar diffusion test

[0081] level Degree of reaction Reaction area observation 0 none No reaction area was observed around or under the sample. 1 slight There are some deformed or degenerated cells under the sample 2 Mild The reaction area is limited to the area below the sample 3 Moderate The reaction area exceeds the sample size to 1.0 cm 4 Severe The reaction area exceeds the sample by more than 1.0 cm

[0082] 3. Experimental results

[0083] The experimental results are shown in Table 2 below.

[0084] Table 2: Results of cytotoxicity test in agar diffusion assay

[0085]

[0086] From the experimental results in Table 2, it can be seen that the cytotoxicity grades of the dressings of Example 1 and Example 2 were both grade 1, while those of Comparative Example 1 and Comparative Example 2 were grade 2, which indicates that the combination of fish skin protein short peptide HPLGL and modified β-lactoglobulin may reduce the cytotoxicity of the latter.

[0087] Effect Example 2: Experimental study on the anti-HPV effect of the functional gynecological dressing of the present invention

[0088] The anti-HPV virus experiment was divided into 6 groups, namely, the control group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. The samples prepared in the above Examples 1-2 and Comparative Examples 1-3 were used for the experiment, and the control group used a matrix component without active ingredients, prepared in the same way.

[0089] Example 2 A quantitative inactivation experiment of HPV16 and HPV18 suspensions was used to determine the anti-HPV virus effect of the functional gynecological dressing of the present invention.

[0090] Vero-E6 cells (provided by the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured and used for virus experiments when they were fully grown. The experimental viruses HPV16 and HPV18 were diluted 10 times with cell culture medium and then inoculated into cell bottles that were fully grown with monolayer cells and placed in a 37°C incubator to allow the viruses to adsorb and grow on the cells.

[0091] Observe the lesions daily, and harvest the virus when 3 / 4 of the cells show lesions. In an ice bath, use ultrasound to break the host cells and release the virus in the culture medium containing the virus and host cells. Then, quickly centrifuge at 6000rpm for 15 minutes to remove the cell debris precipitate, and the supernatant is the required virus suspension. Dispense 1.0mL per tube into 1.5mL sterile centrifuge tubes. For each virus, take 1 virus suspension and determine its virus titer according to the virus titration method.

[0092] Take the dressing sample to be tested and dilute it with sterile water to 1.25 times the concentration of the original solution, and place it in a 20℃ water bath for use. Take 200μL of each virus stock solution and 800μL of the sample dilution to be tested in a 20℃ water bath for 10min, mix it immediately and record the time. After the specified time, take out 100μL immediately, add it to the neutralizer and mix it. Each group carried out virus titer determination, first use cell maintenance culture medium to make a 10-fold serial dilution of the titration sample, and then titrate the residual virus in each dilution sample on a 96-well culture plate, and make 4 wells for each dilution (each well should be full of monolayer host cells), and place it at 37℃ for 1h~2h to ensure that all residual viruses are adsorbed on the cells. Take out the culture plate and replace the cell maintenance culture medium. Continue to place it in a carbon dioxide incubator (37℃, 5% CO2) for culture, observe the cell lesions under a microscope every day, observe for 3 consecutive days, and observe and record the cell lesions in each well. The test results are shown in Table 3 below.

[0093] Table 3: HPV virus killing test results of each group of samples

[0094]

[0095]

[0096] The experimental results in Table 3 show that the samples of Example 1 and Example 2 have very good virus killing effects on HPV16 and HPV18, and their antiviral activity rates are all >99.99%. Combining the results of Comparative Example 1 to Comparative Example 3, it can be seen that the fish skin protein short peptide HPLGL alone has no antiviral effect on HPV16 and HPV18, but the combination of fish skin protein short peptide HPLGL and modified β-lactoglobulin can significantly enhance the antiviral effect of modified β-lactoglobulin on HPV16 and HPV18. It can be seen that the combination of modified β-lactoglobulin and fish skin protein short peptide HPLGL has a significant synergistic effect in anti-HPV virus.

[0097] Effect Example 3: Antibacterial effect experiment of the functional gynecological dressing of the present invention

[0098] The antibacterial experiment was divided into 6 groups, namely, the control group, Example 1 group, Example 2 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. The samples prepared by the above Examples 1-2 and Comparative Examples 1-3 were used for the experiment, and the control group used a matrix component without active ingredients, prepared in the same way.

[0099] Each group of samples was diluted 2 times with PDA medium, and 1 mL was added to 50°C PDA medium and poured into a culture dish to obtain a PDA plate for sample treatment. Use an inoculation loop to pick the strains of Staphylococcus aureus and Escherichia coli on the slope of the test tube and put them in sterile water with glass beads at a concentration of 106 cfu / mL, shake, filter and prepare suspensions of Staphylococcus aureus and Escherichia coli respectively as test strains. Staphylococcus aureus and Escherichia coli strains were activated on PDA flat culture medium without sample treatment, and the pathogen cakes to be used cultured on PDA culture medium were cut with a hole puncher, and the mycelium was inoculated in the center of the solidified PDA culture medium containing the sample with the mycelium facing up, and placed in the dark at 25°C. When the colony diameter of the control group grows to be larger than 2 / 3 of the diameter of the culture dish, the colony diameter (mm) was measured with a caliper. The diameter of each colony was measured vertically once by the cross method, and the average value was taken; each treatment was repeated 3 times, and the growth inhibition rate of each test group sample on colony expansion was calculated by comparison with the control group. The results of the antibacterial experiment are shown in Table 4. Inhibition rate = (control group colony diameter - drug treatment colony diameter) / control group colony diameter × 100%.

[0100] Table 4: Antibacterial test results of each group of samples

[0101]

[0102] The experimental results in Table 4 show that the samples of Example 1 and Example 2 have very good antibacterial effects on Staphylococcus aureus and Escherichia coli. Combining the results of Comparative Example 1 to Comparative Example 3, it can be seen that the antibacterial effect of the dressing samples of the present invention may be mainly produced by the fish skin protein short peptide HPLGL, and the combination of modified β-lactoglobulin and fish skin protein short peptide HPLGL produces a significant synergistic effect in antibacterial effect.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A functional gynecological dressing, characterized in that: The functional gynecological dressing comprises the following components in percentage by mass: 0.03% to 0.04% modified β-lactoglobulin, 0.01% to 0.02% fish skin protein short peptide, 3% glycerol, 5% anhydrous ethanol, and 0.3% carbomer, wherein the weight ratio of the modified β-lactoglobulin to the fish skin protein short peptide is 4:1 or 3:2, the amino acid sequence of the fish skin protein short peptide is HPLGL (His-Pro-Leu-Gly-Leu), the modified β-lactoglobulin is β-lactoglobulin modified with an active acid anhydride, and the active acid anhydride is 3-hydroxy-phthalic anhydride.

2. The functional gynecological dressing according to claim 1, characterized in that: The modification method comprises the following steps: adding a saturated active anhydride solution to a β-lactoglobulin aqueous solution in batches, reacting the saturated active anhydride solution each time, and then adjusting the pH to 5-9; dialyzing the anhydride-treated β-lactoglobulin solution with a phosphate buffer for 3-5 times to obtain modified β-lactoglobulin, wherein the saturated active anhydride solution is a saturated dimethyl sulfoxide solution of 3-hydroxy-phthalic anhydride, and the pH regulator is NaOH, Ca(OH)2, KOH, Na3PO4, ammonium hydroxide, triethanolamine or ethylenediamine.

3. The functional gynecological dressing according to claim 1, characterized in that: The fish skin protein short peptide is a tilapia skin enzymatic hydrolysate or is prepared by a solid phase synthesis method.

4. Use of the functional gynecological dressing according to any one of claims 1 to 3 in the preparation of a medicament for treating HPV16 or HPV18 infection.

5. The use according to claim 4, characterized in that: The drug also has an effect against Staphylococcus aureus or Escherichia coli.

Images