Gynecological gel containing recombinant mussel mucin as well as preparation method and application of gynecological gel

Abstract

The invention relates to the technical field of gynecological gel, and discloses gynecological gel containing recombinant mussel mucin as well as a preparation method and application of the gynecological gel. The gynecological gel containing the recombinant mussel mucin comprises the following raw material components in percentage by mass: 0.01%-50% of the recombinant mussel mucin, 0.1%-20% of a gel matrix, 0.01%-1.0% of a pH regulator, 0.05%-1% of a preservative, 0.5%-20% of a humectant, 10%-50% of a protein protective agent and the balance of water, the gel matrix comprises a thickening agent and a suspending aid, and the protein protective agent is obtained by dissolving a dispersing agent, a chelating agent and a protein stabilizer in a buffer solution. The gynecological gel provided by the invention has good film-forming property, forms a protective gel film on a vaginal wall, physically isolates the vaginal wall from external bacteria, is used for protecting gynecological mucosa and preventing colonization of pathogenic microorganisms, has good anti-inflammatory and anti-oxidation effects, and can be used for preventing or treating gynecological diseases.

Description

Technical Field

[0001] The invention relates to the technical field of gynecological gel, in particular to a gynecological gel containing recombinant mussel mucin and a preparation method and application thereof. Background Art

[0002] In today's society, gynecological health has become an important issue concerning women's well-being and quality of life. The widespread, complex and high incidence of gynecological diseases not only profoundly affect women's physical and mental health, but also highlight the urgent need for society to pay in-depth attention to and innovate in this field.

[0003] Although traditional methods such as drug therapy, physical therapy and surgical treatment can effectively control and treat common gynecological diseases such as vaginitis, cervicitis, pelvic inflammatory disease, etc. to a certain extent, they also have some limitations that cannot be ignored. For example, long-term or improper use of antibiotics and hormone drugs may lead to side effects such as increased drug resistance and imbalance of hormone levels. In addition, oral or injectable medications require long-term patient persistence, while local administration methods such as vaginal suppositories may lead to poor patient compliance due to inconvenience or discomfort. More importantly, some local administration methods may not ensure that the drug reaches an effective concentration at the site of the lesion, thus affecting the treatment effect.

[0004] In addition, as an innovative form of topical medication, gynecological gel products currently on the market still face some challenges in terms of stability, mainly manifested in gel stratification causing the product to become uneven, pH value changes, and active ingredients degradation during storage, etc., which will lead to unstable appearance and efficacy of the product, and ultimately affect the product's use effect and safety.

[0005] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a gynecological gel containing recombinant mussel mucin and a preparation method and application thereof, aiming to solve the problems of the existing gynecological gel lacking stability and poor efficacy.

[0007] The technical solution of the present invention is as follows:

[0008] The first aspect of the present invention provides a gynecological gel containing recombinant mussel mucin, comprising the following raw material components in percentage by mass: 0.01%-50% recombinant mussel mucin, 0.1%-20% gel matrix, 0.01%-1.0% pH regulator, 0.05%-1% preservative, 0.5%-20% moisturizer, 10%-50% protein protectant, and the remainder water; the gel matrix comprises a thickener and a suspending agent, and the protein protectant is obtained by dissolving a dispersant, a chelating agent and a protein stabilizer in a buffer solution.

[0009] Optionally, in the gel matrix, the mass ratio of the thickener to the suspending agent is (0.05-3):(0.05-5).

[0010] Optionally, the thickener is one or more of carbomer, alginate, gelatin, pectin, carrageenan, cationic guar gum, chitin, ethylene polymer and acrylic resin.

[0011] Optionally, the suspending agent is one or more of polyvinyl alcohol, xanthan gum, methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.

[0012] Optionally, the mass ratio of the dispersant, the chelating agent, the protein stabilizer and the buffer solution is (0.5-20):(0.01-1):(0.01-3.0):(10-50).

[0013] Optionally, the dispersant is one or more of glycerol, propylene glycol, polyethylene glycol and butylene glycol.

[0014] Optionally, the chelating agent is one of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylene glycol tetraacetate, trisodium aminotriacetate, pentasodium diethylenetriaminepentaacetate, citric acid, tartaric acid, gluconic acid, sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate.

[0015] Optionally, the protein stabilizer is one or more of ergothioneine, sodium pyrosulfite, sodium dodecylbenzene sulfonate and lipoic acid.

[0016] Optionally, the buffer solution is one or more of acetate buffer, citrate buffer, PBS buffer, phosphoric acid and its salt solution, Tris buffer, and malic acid.

[0017] Optionally, the pH regulator is one or more of triethanolamine, hydrochloric acid, tromethamine, sodium hydroxide, sodium bicarbonate and sodium dihydrogen phosphate.

[0018] Optionally, the preservative is one or more of sodium benzoate, phenoxyethanol, methylparaben, sodium methylparaben, sodium ethylparaben and sodium propylparaben.

[0019] Optionally, the humectant is one or more of xylitol, hexylene glycol, polypropylene glycol and sorbitol.

[0020] The second aspect of the present invention provides a method for preparing the gynecological gel containing recombinant mussel mucin, comprising the steps of:

[0021] The suspending agent, thickener and water are mixed, heated and stirred, and after homogenization, a pH adjuster is added to adjust the pH value to 4.5-5.5 to obtain a gel matrix;

[0022] A dispersant, a chelating agent, a protein stabilizer and a buffer solution are prepared to obtain a protein protective agent, and a recombinant mussel mucin solution or a lyophilized powder is added to the protein protective agent to mix, so as to obtain a protein solution system containing the recombinant mussel mucin;

[0023] The protein solution system is added into the gel matrix, stirred, a moisturizer, a preservative and a pH adjuster are added, and the pH value is adjusted to 3.5-7.5 to obtain the gynecological gel containing recombinant mussel mucin.

[0024] Optionally, the concentration of the recombinant mussel mucin solution is 5 mg / g-100 mg / g.

[0025] The third aspect of the present invention provides a use of the gynecological gel containing recombinant mussel mucin in the preparation of drugs for preventing or treating gynecological diseases.

[0026] Beneficial effects:

[0027] 1. Since recombinant mussel mucin has the function of promoting cell adhesion, crawling and growth, the gynecological gel containing recombinant mussel mucin provided by the present invention helps to accelerate the repair of damaged areas of the vaginal wall and maintain the integrity and health of the vaginal mucosa; based on the excellent anti-inflammatory effect of recombinant mussel mucin, the gynecological gel containing recombinant mussel mucin helps to promote the repair and healing of damaged tissues, reduce vaginal inflammation, and relieve symptoms such as redness, swelling and itching when the vagina is damaged or inflamed; based on the excellent antioxidant effect of recombinant mussel mucin, the gynecological gel containing recombinant mussel mucin can neutralize some harmful free radicals, protect vaginal cells from damage by oxidative stress, and thus maintain vaginal health.

[0028] 2. The present invention adopts a protein protective agent composed of the dispersant, chelating agent, protein stabilizer and buffer, and the protein protective agent can have multiple synergistic effects with the recombinant mussel mucin, giving the gynecological gel containing the recombinant mussel mucin excellent moisturizing performance, vaginal mucosa repair and care, anti-inflammatory and antioxidant effects. The protein protective agent is beneficial to the stability of the recombinant mussel mucin and DOPA in the gynecological gel, so that the gynecological gel containing the recombinant mussel mucin can be stable and exert its efficacy.

[0029] 3. The gynecological gel containing recombinant mussel mucin provided by the present invention has good film-forming properties and can form a protective gel film on the vaginal wall to physically isolate the vaginal wall from external bacteria, and is used for gynecological mucosal protection to prevent the colonization of pathogenic microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a comparison photo of the gynecological gel containing recombinant mussel mucin of Example 5 of the present invention and the blank control group in a human keratinocyte migration experiment.

[0031] Figure 2 This is a comparison chart of the test results of the gynecological gel containing recombinant mussel mucin in Example 5 of the present invention, the blank control group and the positive control group in the human keratinocyte migration experiment.

[0032] Figure 3 This is a comparison chart of the test results of the gynecological gel containing recombinant mussel mucin of Example 5 of the present invention and the blank control group in a human fibroblast adhesion experiment.

[0033] Figure 4 This is a graph showing the test results of TNF-α content in the gynecological gel containing recombinant mussel mucin of Example 5 of the present invention, the negative control group, the positive control group and the blank control group.

[0034] Figure 5 This is a graph showing the test results of IL-6 content in the gynecological gel containing recombinant mussel mucin in Example 5 of the present invention, the negative control group, the positive control group and the blank control group.

[0035] Figure 6 The figure shows the change in vaginal cleanliness of the gynecological gel containing recombinant mussel mucin according to Example 5 of the present invention after continuous use by subjects for 14 days.

[0036] Figure 7 The figure shows the change in pH of vaginal secretions after subjects used the gynecological gel containing recombinant mussel mucin according to Example 5 of the present invention continuously for 14 days.

[0037] Figure 8 The figure shows the changes in vaginal flora of subjects who used the gynecological gel containing recombinant mussel mucin according to Example 5 of the present invention continuously for 14 days.

[0038] Fig. 9 The figure shows the changes in vaginal inflammation of subjects after continuous use of the gynecological gel containing recombinant mussel mucin according to Example 5 of the present invention for 14 days. DETAILED DESCRIPTION

[0039] The present invention provides a gynecological gel containing recombinant mussel mucin and a preparation method and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0041] As an innovative form of topical medication, gynecological gel brings new hope to the treatment of gynecological diseases with its convenience and direct local effect. Compared with traditional treatment methods, gynecological gel can directly act on the lesion site to form a drug protection layer, thereby increasing the local drug concentration and enhancing the therapeutic effect. Since the drug mainly acts locally, the risk of systemic side effects is also reduced accordingly. The lubricity and comfort of gynecological gel make it easy to use, which not only improves patient acceptance, but also significantly improves treatment compliance. In addition, the gel dosage form can be designed as a sustained-release or controlled-release system to enable the drug to be continuously released over a certain period of time, reduce the number of dosing times, improve the therapeutic effect, and reduce the economic and psychological burden on patients. The versatility of gynecological gel is also reflected in the fact that in addition to its therapeutic effect, it can also add lubricating, moisturizing, antibacterial and other ingredients to provide comprehensive care for patients. At the same time, the foot silk glands of mussels can secrete a very special protein with super strong viscosity, which can make mussels firmly adhere to objects in any environment, called "mussel mucin". In clinical trials, mussel mucin has been shown to form a mesh biofilm covering the skin surface, helping the skin resist external stimuli and avoid microbial invasion. It also has strong anti-inflammatory and antioxidant capabilities, and can accelerate the repair and healing of damaged parts of the skin surface. Therefore, the inventors thought of applying mussel mucin to gel to obtain a gynecological gel containing recombinant mussel mucin that has both the dual effects of mussel mucin and gel formulation.

[0042] The embodiment of the present invention provides a gynecological gel containing recombinant mussel mucin, comprising the following raw material components in percentage by mass: 0.01%-50% of recombinant mussel mucin, 0.1%-20% of gel matrix, 0.01%-1.0% of pH regulator, 0.05%-1% of preservative, 0.5%-20% of moisturizer, 10%-50% of protein protectant and the remainder of water; the gel matrix comprises a thickener and a suspending agent, and the protein protectant is obtained by dissolving a dispersant, a chelating agent and a protein stabilizer in a buffer solution.

[0043] The gynecological gel containing recombinant mussel mucin provided by the present invention has good film-forming property, can form a protective gel film on the vaginal wall, physically isolate the vaginal wall from external bacteria, and is used for gynecological mucosal protection to prevent pathogenic microorganisms from colonizing. At the same time, it has good anti-inflammatory and antioxidant effects. Recombinant mussel mucin is rich in positively charged amino acids and DOPA groups, wherein the DOPA group is a key factor in the adhesion ability of mussel mucin, and the DOPA group also has the ability to resist inflammation, resist oxidation and promote cell adhesion. The DOPA group is an amino acid containing a catechol group, which is extremely unstable in the natural environment and is prone to self-oxidation reaction to generate dopamine-quinone. Moreover, the protein protective agent is beneficial to the stability of the recombinant mussel mucin, and the polyol dispersant, chelating agent, protein stabilizer and recombinant mussel mucin have multiple synergistic effects, which endow the gynecological gel containing recombinant mussel mucin with excellent moisturizing performance, vaginal mucosal repair and care, anti-inflammatory and antioxidant effects.

[0044] In the gel matrix, the mass ratio of thickener to suspending agent is (0.05-3): (0.05-5). The gynecological gel prepared under this ratio has moderate viscosity, does not flow, has a wide range of effects, has the common advantages of liquid dosage forms and solid dosage forms, and is conducive to the suspension stability of recombinant mussel mucin.

[0045] In some specific embodiments, in the gel matrix, the mass ratio of the thickener to the suspending agent is (0.05-3):(0.05-5).

[0046] The present invention adopts the gel matrix within the ratio range to make the gynecological gel containing recombinant mussel mucin moderate in viscosity, non-flowing, and having a wide range of effects, having the common advantages of liquid dosage forms and solid dosage forms, and at the same time being conducive to the suspension stability of the recombinant mussel mucin.

[0047] The gel matrix provided by the present invention is prepared by mixing a thickener and a suspending agent in a specific ratio. The presence of the suspending agent is beneficial to improving the stability of the gel. The suspending agent in the above-mentioned ratio range is beneficial to improving the formability and spreadability of the gel.

[0048] In some specific embodiments, the thickener is one or more of carbomer, alginate, gelatin, pectin, carrageenan, cationic guar gum, chitin, ethylene polymer and acrylic resin.

[0049] In some specific embodiments, the suspending agent is one or more of polyvinyl alcohol, xanthan gum, methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.

[0050] In some specific embodiments, the mass ratio of the dispersant, the chelating agent, the protein stabilizer and the buffer solution is (0.5-20): (0.01-1): (0.01-3.0): (10-50).

[0051] When the mass ratio of the dispersant, the chelating agent, the protein stabilizer and the buffer solution is within this range, the activity and properties of the recombinant mussel mucin can be effectively stabilized while ensuring that DOPA is not easily oxidized.

[0052] In some specific embodiments, the dispersant is one or more of glycerol, propylene glycol, polyethylene glycol and butylene glycol.

[0053] The dispersant used in the present invention is mainly a polyol, which has excellent moisturizing properties. The synergistic effect of the polyol and the recombinant mussel mucin, in the presence of hydroxyl free radicals, the gel containing the recombinant mussel mucin exhibits a low carbonyl content and a high thiol content, which can effectively improve the protein stability.

[0054] In some specific embodiments, the chelating agent is one of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid, disodium ethylenediaminetetraacetate, tetrasodium ethylene glycol tetraacetate, trisodium aminotriacetate, pentasodium diethylenetriaminepentaacetate, citric acid, tartaric acid, gluconic acid, sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate.

[0055] The chelating agent used in the present invention can effectively complex metal ions to prevent potential damage to the protein structure by metal ions. In addition, it can maintain the three-dimensional conformation of the protein, stabilize free sulfhydryl groups and reduce the protein. The addition of the chelating agent can significantly improve the stability of the recombinant mussel mucin, thereby ensuring its biological activity and therapeutic effect.

[0056] In some specific embodiments, the protein stabilizer is one or more of ergothioneine, sodium pyrosulfite, sodium dodecylbenzene sulfonate and lipoic acid.

[0057] The protein stabilizer used in the present invention contains reducing groups such as disulfide bonds, which can effectively remove active oxygen free radicals, protect proteins from attacks by oxidants, and maintain the structure and function of proteins. At the same time, the protein stabilizer can also block the chain reaction of protein oxidation, inhibit the formation of carbonyl groups, and reduce protein carbonylation. Therefore, the protein stabilizer can effectively protect the recombinant mussel mucin and the DOPA groups contained therein to prevent them from being oxidized, thereby ensuring the stability of the recombinant mussel mucin and DOPA.

[0058] In some specific embodiments, the buffer solution is one or a combination of acetate buffer, citrate buffer, PBS buffer, phosphate and its salts, Tris buffer, and malic acid.

[0059] The present invention uses a buffer solution to maintain the pH value of the system within a certain range, preventing changes in protein structure and loss of activity caused by pH fluctuations. Since the dopa group contained in the recombinant mussel mucin is sensitive to pH, it is very important to maintain the pH value of the system stable. Adding a buffer solution can resist small acid-base changes and keep the pH value relatively stable, thereby protecting the recombinant mussel mucin and the dopa group.

[0060] The protein protective agent provided by the present invention is not only beneficial to the stability of the protein, but also beneficial to the stability of the gel matrix. The polyol dispersant contained in the protein protective agent can be used as a hydroxyl donor to form hydrogen bonds with the carboxyl group (-COOH) in the gel network, which is beneficial to improve the formation of a denser gel network structure inside the gel, thereby improving the stability of the gel matrix; in addition, the enhanced gel network structure helps to improve the water holding capacity and mechanical properties of the gel, making it more stable during use. The chelating agent contained in the protein protective agent can effectively chelate the metal ions of the system, reduce the oxidation reaction catalyzed by the metal ions, and protect the molecules in the gel from oxidation. The buffer solution contained in the protein protective agent is beneficial to stabilize the pH of the system, and then stabilize the performance of the gel system, such as thickening effect, rheology, chemical stability, etc.

[0061] In some specific embodiments, the preservative is one or more of sodium benzoate, phenoxyethanol, methylparaben, sodium methylparaben, sodium ethylparaben and sodium propylparaben.

[0062] In some specific embodiments, the humectant is one or more of xylitol, hexylene glycol, polypropylene glycol and sorbitol.

[0063] In some specific embodiments, the pH adjuster is one or more of triethanolamine, hydrochloric acid, tromethamine, sodium hydroxide, sodium bicarbonate and sodium dihydrogen phosphate.

[0064] The second aspect of the present invention provides a method for preparing the gynecological gel containing recombinant mussel mucin, comprising the steps of:

[0065] The suspending agent, thickener and water are mixed, heated and stirred, and after homogenization, a pH adjuster is added to adjust the pH value to 4.5-5.5 to obtain a gel matrix;

[0066] A dispersant, a chelating agent, a protein stabilizer and a buffer solution are prepared to obtain a protein protective agent, and a recombinant mussel mucin solution or a lyophilized powder is added to the protein protective agent to mix, so as to obtain a protein solution system containing the recombinant mussel mucin;

[0067] The protein solution system is added into the gel matrix, stirred, a moisturizer, a preservative and a pH adjuster are added, and the pH value is adjusted to 3.5-7.5 to obtain the gynecological gel containing recombinant mussel mucin.

[0068] The preparation process of the gynecological gel containing recombinant mussel mucin in the present invention is simple. The gynecological gel containing recombinant mussel mucin is subsequently degassed and loaded into a syringe for packaging, and finally irradiated and sterilized to obtain a product for commercial promotion.

[0069] In some specific embodiments, the concentration of the recombinant mussel mucin solution is 5 mg / g-100 mg / g.

[0070] The third aspect of the present invention provides a use of the gynecological gel containing recombinant mussel mucin in the preparation of drugs for preventing or treating gynecological diseases. The gynecological diseases are bacterial vaginitis, vulvovaginal candidiasis, cervicitis, cervical erosion, vaginal itching, vaginal odor, vaginal pain, vaginal congestion and edema, repair of damaged vagina or abnormal leucorrhea. At the same time, the gynecological gel containing recombinant mussel mucin can also be used for postpartum repair, menopausal symptom relief, postoperative care for gynecological surgery, daily private care, anti-aging maintenance, cesarean section scar repair, vaginal microecological balance, promotion of mucosal repair, post-sexual repair, etc.

[0071] The following describes it in detail through specific embodiments.

[0072] Example 1

[0073] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials in terms of mass percentage: 5% gel matrix, 20% protein protective agent, 0.1% methylparaben, 20% 5mg / g recombinant mussel mucin solution, 0.1% sorbitol, 0.1% tromethamine, and the balance purified water.

[0074] The gel matrix is ​​composed of a thickener (Carbomer 940) and a suspending agent (hydroxyethyl cellulose) in a mass ratio of 2:3.

[0075] The protein protective agent is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:0.1:20, wherein the dispersant is composed of propylene glycol and polyethylene glycol 400 in a mass ratio of 1:1, the chelating agent is disodium ethylenediaminetetraacetate, the protein stabilizer is ergothioneine, and the buffer solution is an acetate buffer.

[0076] The preparation steps of the gynecological gel containing recombinant mussel mucin are as follows:

[0077] S1. Accurately weigh the thickener and suspending agent, add purified water, start heating and stirring, add the thickener and suspending agent in sequence under stirring conditions (60r / min), stir for 30 minutes and then start homogenization (5min), after the homogenization is completed, add tromethamine under stirring conditions to make the pH of the gel matrix 5.0.

[0078] S2. Accurately weigh the above dispersant, chelating agent, protein stabilizer and buffer, add them in sequence under stirring and mix them thoroughly;

[0079] S3, accurately weigh the above recombinant mussel mucin solution, add it to the protein protective agent obtained in S2, and stir and mix thoroughly for 10 minutes;

[0080] S4. Slowly add the solution obtained in S3 to the gel matrix obtained in S1 under stirring, and add a moisturizer, a preservative and a pH adjuster in sequence after thorough mixing, so that the pH of the obtained recombinant mussel mucin gel is 5.8.

[0081] S5. Finally, the obtained recombinant mussel mucin gel is vacuum-defoamed, then inner-wrapped and outer-wrapped, and then terminally sterilized.

[0082] Example 2

[0083] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials in terms of mass percentage: 10% gel matrix, 10% protein protective agent, 0.1% phenoxyethanol, 20% 5 mg / g recombinant mussel mucin solution, 0.1% sorbitol, 0.2% tromethamine, and the balance purified water.

[0084] The gel matrix is ​​composed of carbomer U20 and methylcellulose in a mass ratio of 5:0.05.

[0085] The protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:0.1:10, wherein the dispersant is composed of propylene glycol and polyethylene glycol 400 in a mass ratio of 1:1, the chelating agent is disodium ethylenediaminetetraacetate, the protein stabilizer is sodium pyrosulfite, and the buffer solution is an acetate buffer.

[0086] The preparation method of the gynecological gel containing recombinant mussel mucin is the same as that in Example 1.

[0087] Example 3

[0088] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials in terms of mass percentage: 5% gel matrix, 10% protein protective agent, 0.1% phenoxyethanol, 20% 5mg / g recombinant mussel mucin solution, 0.5% sorbitol, 0.15% tromethamine, and the balance purified water.

[0089] The gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 3:1.

[0090] The protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 0.5:0.01:0.01:20, wherein the dispersant is 1,3-propylene glycol, the chelating agent is amino acid acetic acid, the protein stabilizer is lipoic acid, and the buffer solution is a citric acid buffer solution.

[0091] The preparation method of the gynecological gel containing recombinant mussel mucin is the same as that in Example 1.

[0092] Example 4

[0093] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials, measured by mass percentage: 10% gel matrix, 15% protein protective agent, 0.1% phenoxyethanol, 20% 5mg / g recombinant mussel mucin solution, 1.0% xylitol, 0.45% triethanolamine, and the balance purified water.

[0094] The gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 3:1.

[0095] The protein protective agent is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 10:0.5:2:20, wherein the dispersant is composed of propylene glycol, polyethylene glycol 400 and glycerol in a mass ratio of 3:3:4, the chelating agent is composed of aminotriacetic acid and disodium ethylenediaminetetraacetate in a mass ratio of 1:1, the protein stabilizer is composed of ergothioneine, sodium pyrosulfite and sodium dodecylbenzenesulfonate in a mass ratio of 0.1:0.1:2, and the buffer is a citric acid buffer.

[0096] The preparation method of the gynecological gel containing recombinant mussel mucin is the same as that in Example 1.

[0097] Example 5

[0098] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials in terms of mass percentage: 8% gel matrix, 30% protein protective agent, 0.1% sodium benzoate, 2% 50mg / g recombinant mussel mucin solution, 5.0% sorbitol, 0.4% triethanolamine, and the balance purified water.

[0099] The gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 3:1.

[0100] The protein protective agent is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:0.1:20, wherein the dispersant is composed of propylene glycol and polyethylene glycol 400 in a mass ratio of 1:1, the chelating agent is disodium ethylenediaminetetraacetate, the protein stabilizer is sodium pyrosulfite, and the buffer is Tris buffer.

[0101] The preparation method of the gynecological gel containing recombinant mussel mucin is the same as that in Example 1.

[0102] Example 6

[0103] This embodiment provides a gynecological gel containing recombinant mussel mucin. The gynecological gel containing recombinant mussel mucin comprises the following raw materials in terms of mass percentage: 15% gel matrix, 20% protein protective agent, 0.05% sodium methylparaben, 0.05% sodium propylparaben, 0.1% recombinant mussel mucin freeze-dried powder, 5.0% sorbitol, 0.4% triethanolamine, and the balance purified water.

[0104] The gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 3:1.

[0105] The protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:0.1:40, wherein the dispersant is composed of propylene glycol and polyethylene glycol 400 in a mass ratio of 1:1, the chelating agent is disodium ethylenediaminetetraacetate, the protein stabilizer is sodium pyrosulfite, and the buffer is an acetate buffer.

[0106] The preparation method of the gynecological gel containing recombinant mussel mucin is the same as that in Example 1.

[0107] Comparative Example 1

[0108] Compared with Example 5, the only difference is that in the gynecological gel containing recombinant mussel mucin, the gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 0.04:1.

[0109] Comparative Example 2

[0110] Compared with Example 5, the only difference is that in the gynecological gel containing recombinant mussel mucin, the gel matrix is ​​composed of carbomer 941 and methyl cellulose in a mass ratio of 0.6:6.

[0111] Comparative Example 3

[0112] Compared with Example 5, the difference is that in the gynecological gel containing recombinant mussel mucin, the dispersant, chelating agent, protein stabilizer and buffer solution are composed of a mass ratio of 0.4:0.1:0.1:20, and the dispersant is composed of propylene glycol and glycerol in a mass ratio of 1:1.

[0113] Comparative Example 4

[0114] Compared with Example 5, the difference is that in the gynecological gel containing recombinant mussel mucin, the protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:4:20, and the dispersant is composed of propylene glycol and glycerol in a mass ratio of 1:1.

[0115] Comparative Example 5

[0116] Compared with Example 5, the difference is that in the gynecological gel containing recombinant mussel mucin, the protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:1.5:0.1:20, and the dispersant is composed of propylene glycol and glycerol in a mass ratio of 1:1.

[0117] Comparative Example 6

[0118] Compared with Example 5, the difference is that in the gynecological gel containing recombinant mussel mucin, the protein protectant is composed of a dispersant, a chelating agent, a protein stabilizer and a buffer solution in a mass ratio of 6:0.1:0.005:20, and the dispersant is composed of propylene glycol and glycerol in a mass ratio of 1:1.

[0119] Comparative Example 7

[0120] Compared with Example 5, the difference is that the gynecological gel containing recombinant mussel mucin does not contain a protein protective agent.

[0121] The preparation method of the gynecological gel containing recombinant mussel mucin is as follows:

[0122] S1. Accurately weigh the thickener and suspending agent, add purified water, start heating and stirring, add methylcellulose and carbomer in sequence under stirring conditions (60r / min), stir for 30 minutes and then start homogenization (5 minutes), after the homogenization is completed, add tromethamine under stirring conditions to adjust the pH of the gel matrix to 5.5.

[0123] S2. Accurately weigh the recombinant mussel mucin solution, slowly add it to the gel matrix obtained in S1 under stirring, and stir and mix thoroughly for 30 minutes;

[0124] S3. Add a moisturizer, a preservative and a pH adjuster in sequence under stirring to adjust the pH of the obtained recombinant mussel mucin gel to 5.5.

[0125] S4. Finally, the obtained recombinant mussel mucin gel is vacuum-defoamed, then inner-wrapped and outer-wrapped, and then terminally sterilized.

[0126] The gynecological gel containing recombinant mussel mucin prepared in Examples 1-6 of the present invention and Comparative Examples 1-7 was tested for performance, and the results were as follows:

[0127] (1) Gel key indicators

[0128] 1.1 Evaluation of gel state: The gynecological gel containing recombinant mussel mucin obtained in the above-mentioned examples and comparative examples was placed at 60°C for accelerated aging for 14 days, and then taken out. The samples after accelerated aging and the samples without accelerated aging were squeezed onto a blank culture dish at the same time to observe the gel state. The results are shown in Table 2.

[0129] 1.2 Gel color evaluation: The gynecological gel containing recombinant mussel mucin obtained in the examples and comparative examples was placed at 60°C for accelerated aging for 14 days, and then taken out after the accelerated aging. The samples after accelerated aging and the samples without accelerated aging were squeezed onto a blank culture dish at the same time, and the color changes of each group before and after aging were compared. The results are shown in Table 2.

[0130] 1.3 Protein content determination: The finished recombinant mussel mucin gynecological gel obtained in the embodiment and the comparative example was placed at 60°C for accelerated aging for 14 days, and then taken out after the accelerated aging. The recombinant mussel mucin gynecological gel after accelerated aging was taken, and the gel was squeezed out as a test sample, and tested at room temperature according to the fourth part of the 2020 edition of the Chinese Pharmacopoeia (General Rule 0731 Protein Content Determination Method Second Method Folin Phenol Method (Lowry Method)). The results are shown in Table 2.

[0131] 1.4 Determination of DOPA Content The finished recombinant mussel mucin gynecological gel obtained in the examples and comparative examples was placed at 60°C for accelerated aging for 14 days, taken out after the end, and the recombinant mussel mucin gynecological gel after accelerated aging was taken, and the gel was squeezed out as a test sample, and the DOPA content test in Appendix B of YY / T1293.6-2020 "Contact Wound Dressings Part 6: Mussel Mucin Dressings" was tested. The results are shown in Table 2.

[0132] Table 2

[0133]

[0134]

[0135] It can be seen from the test results in Table 2 above that within the content range described in the formula, different thickeners, suspending agents, dispersants, chelating agents, protein stabilizers and their contents and recombinant mussel mucins of different dosage forms can achieve a good gel state, and the color, protein content and DOPA content of recombinant mussel mucins are stable. When the proportion of thickener in the gel matrix is ​​lower than the formula content range, the viscosity of the prepared gynecological gel will be low, the gel state will deteriorate, and the gel will become thinner, which will cause the use process to flow or flow out easily, resulting in poor use effect; when the proportion of suspending agent in the gel matrix is ​​too high and the proportion of thickener is low, the gel continuity will be poor, slight discontinuity will occur, and some protein will precipitate; when the mass proportion of dispersant is lower than the formula protection range, the gel uniformity is poor, and a small part of the protein is analyzed out, showing an unstable state; when the content of each component in the protein protective agent system is outside the formula protection range, the gynecological gel containing recombinant mussel mucin will have yellowing of protein, precipitation or unstable protein content and DOPA content, resulting in gel stability, which ultimately affects the efficacy of the gel.

[0136] (2) In vitro cell repair efficacy test

[0137] The gynecological gel containing recombinant mussel mucin is used to carry out cell experiments and culture cells, and its influence on the migration effect and adhesion of human keratinocytes is measured.

[0138] 2.1 Cell migration test method:

[0139] Select well-grown human keratinocytes, digest them with trypsin, dilute them to a suitable concentration, and add them to a 6-well plate. Incubate them in an incubator for 24 hours. After the incubation, check and confirm the cell adhesion. If the requirements are met, add samples. In the experiment, set up a blank control group (control), a positive control group (EGF), and a sample group of recombinant mussel mucin gynecological gel (MGA). Prepare samples of corresponding concentrations in serum-free culture medium. After scratching the 6-well plate, add samples and take photos. Place the cell culture plate in an incubator and incubate for about 24 hours. After the incubation, take photos and analyze the results.

[0140] Figure 1 These are photos of the gynecological gel sample group containing recombinant mussel mucin and the blank control group in the human keratinocyte migration experiment.

[0141] like Figure 2As shown, under the action of gynecological gel containing recombinant mussel mucin, the cell migration effect was significantly improved, and the cell migration rate was increased by 101.3%, indicating that gynecological gel containing recombinant mussel mucin has a promoting effect on the repair of human keratinocytes.

[0142] Among them, *P<0.05, **P<0.01 are the P values ​​obtained by the statistical significance test method, with P<0.05 indicating statistical difference, P<0.01 indicating significant statistical difference, and P<0.001 indicating extremely significant statistical difference, which means that the probability that the difference between samples is caused by sampling error is less than 0.05, 0.01, and 0.001, respectively.

[0143] 2.2 Cell adhesion test method: first prepare samples of corresponding concentrations with PBS, add the samples to a 96-well plate with untreated surface, and place the culture plate in a 4°C refrigerator overnight; discard the samples, select well-grown human fibroblasts (HDF) and culture them in complete culture medium (DMEM + 1% Pen / Strep + 10% FBS). When the cell density reaches 70%-80%, collect the cells and inoculate them in a cell culture plate at a cell density of 20K / well. After culturing in an incubator for 1 hour, aspirate the supernatant, wash twice with PBS, and detect cell viability with CCK-8 reagent.

[0144] like Figure 3 The results showed that under the action of gynecological gel (MGA) containing recombinant mussel mucin, the cell adhesion rate increased by 1101.49%. This shows that MGA has a promoting effect on cell HDF. Among them, ***P<0.0001.

[0145] (3) In vitro anti-inflammatory efficacy test

[0146] 3.1 Test method: Cells were cultured in a 96-well plate. A culture medium containing a certain concentration of gynecological gel (MGA) containing recombinant mucin and LPS was added to the test wells, a cell culture medium containing LPS was added to the negative control wells (NC), a culture medium containing positive control and LPS was added to the positive control wells (PC), and a cell culture medium was added to the blank / solvent control wells (BC). After the addition of samples, the cells were cultured continuously. After 24 hours, the cell supernatant was collected and the TNF-α content was detected.

[0147] like Figure 4 The results showed that after LPS stimulation of cells, the TNF-α content in the experimental group containing recombinant mussel mucin was significantly lower than that in the negative control group. Among them, **P<0.01, ###P<0.001, ***P<0.001.

[0148] 3.2 Test method: Cells were cultured in a 96-well plate, culture medium containing a certain concentration of recombinant mussel mucin gynecological gel (MGA) and LPS was added to the test wells, cell culture medium containing LPS was added to the negative control wells (NC), culture medium containing positive control and LPS was added to the positive control wells (PC), and cell culture medium was added to the blank / solvent control wells (BC). After the addition of samples, the cells were cultured continuously, and the cell supernatant was collected after 24 hours to detect the IL-6 content.

[0149] like Figure 5 The results showed that after LPS stimulation of cells, the IL-6 content in the experimental group containing recombinant mussel mucin was significantly lower than that in the negative control group. **P<0.01.

[0150] (4) Clinical efficacy test for vaginitis

[0151] 20 patients with vaginal inflammation were selected. The cleanliness of vaginal secretions was ≥ Grade II and the patients were enrolled after menstruation was clean for more than 3 days. The product was used for 14 consecutive days according to the product instructions. The product was tested and photographed with vaginal endoscope on the 0th, 3rd, 7th and 14th days, and a combined test for vaginitis was performed. Routine vaginal secretions were tested before and after the test, and the index scores were statistically calculated.

[0152] Vaginitis detection indicators and expression methods:

[0153] a. Normal pH range 3.8-4.5;

[0154] b. The evaluation indexes and descriptions of vaginal cleanliness are shown in Table 3:

[0155] Table 3

[0156]

[0157]

[0158] c. Bacterial flora: The evaluation index and description of the indicator H2O2 are shown in Table 4:

[0159] Table 4

[0160] Evaluation indicators describe "” Grade I, normal flora "±” Grade II, bacterial flora disturbance "+” Grade III, severe abnormal bacterial flora

[0161] d. Inflammation: The evaluation index and description of the indicator - leukocyte esterase (LE) are shown in Table 5:

[0162] Table 5

[0163]

[0164]

[0165] Figures 6 to 9 The results show the changes in vaginal cleanliness, vaginal secretion pH, vaginal flora and vaginal inflammation in subjects who used the gynecological gel containing recombinant mussel mucin continuously for 14 days. Figure 6 It can be seen that after 14 days of continuous use, the subjects' vaginal cleanliness level decreased by 40.5%, and the cleanliness was significantly improved. Figure 7 , Figure 8 and Fig. 9 It can be seen that after 3 consecutive days of use, the pH value of vaginal secretions improved, and the vaginal flora and inflammation conditions improved significantly, with the severity of inflammation decreasing by 25.0% in 3 days; after 14 consecutive days of use, the subjects' vaginal secretion pH and vaginal flora conditions tended to stabilize, and vaginal inflammation conditions continued to improve, with the severity of inflammation decreasing by 43.8% in 14 days, showing significant improvement.

[0166] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A gynecological gel containing recombinant mussel mucin, characterized in that: The invention comprises the following raw material components in percentage by mass: 0.01%-50% of recombinant mussel mucin, 0.1%-20% of gel matrix, 0.01%-1.0% of pH regulator, 0.05%-1% of preservative, 0.5%-20% of moisturizing agent, 10%-50% of protein protective agent and the balance of water; the gel matrix comprises a thickener and a suspending agent, and the protein protective agent is obtained by dissolving a dispersant, a chelating agent and a protein stabilizer in a buffer solution.

2. The gynecological gel containing recombinant mussel mucin according to claim 1, characterized in that: In the gel matrix, the mass ratio of the thickener to the suspending agent is (0.05-3):(0.05-5).

3. The gynecological gel containing recombinant mussel mucin according to claim 2, characterized in that: The thickener is one or more of carbomer, alginate, gelatin, pectin, carrageenan, cationic guar gum, chitin, ethylene polymer and acrylic resin; the suspending agent is one or more of polyvinyl alcohol, xanthan gum, methyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose.

4. The gynecological gel containing recombinant mussel mucin according to claim 1, characterized in that: In the protein protective agent, the mass ratio of the dispersant, the chelating agent, the protein stabilizer and the buffer solution is (0.5-20): (0.01-1): (0.01-3.0): (10-50).

5. The gynecological gel containing recombinant mussel mucin according to claim 4, characterized in that: The dispersant is one or more of glycerol, propylene glycol, polyethylene glycol and butylene glycol; the chelating agent is one of ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid, aminotriacetic acid, diethylenetriaminepentaacetic acid, disodium ethylenediaminetetraacetic acid, tetrasodium ethylene glycol tetraacetic acid, trisodium aminotriacetic acid, pentasodium diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, sodium tripolyphosphate, sodium hexametaphosphate and sodium pyrophosphate; the protein stabilizer is one or more of ergothioneine, sodium pyrosulfite, sodium dodecylbenzenesulfonate and lipoic acid; the buffer solution is one or more of acetate buffer, citric acid buffer, PBS buffer, phosphoric acid and its salt solution, Tris buffer and malic acid.

6. The gynecological gel containing recombinant mussel mucin according to claim 1, characterized in that: The pH regulator is one or more of triethanolamine, hydrochloric acid, tromethamine, sodium hydroxide, sodium bicarbonate and sodium dihydrogen phosphate; and / or the preservative is one or more of sodium benzoate, phenoxyethanol, methylparaben, sodium methylparaben, sodium ethylparaben and sodium propylparaben.

7. The gynecological gel containing recombinant mussel mucin according to claim 1, characterized in that: The moisturizing agent is one or more of xylitol, hexylene glycol, polypropylene glycol and sorbitol.

8. A method for preparing a gynecological gel containing recombinant mussel mucin according to any one of claims 1 to 7, characterized in that: Includes steps: The suspending agent, thickener and water are mixed, heated and stirred, and after homogenization, a pH adjuster is added to adjust the pH value to 4.5-5.5 to obtain a gel matrix; A dispersant, a chelating agent, a protein stabilizer and a buffer solution are prepared to obtain a protein protective agent, and a recombinant mussel mucin solution or a lyophilized powder is added to the protein protective agent to mix, so as to obtain a protein solution system containing the recombinant mussel mucin; The protein solution system is added into the gel matrix, stirred, a moisturizer, a preservative and a pH adjuster are added, and the pH value is adjusted to 3.5-7.5 to obtain the gynecological gel containing recombinant mussel mucin.

9. The method for preparing the gynecological gel according to claim 8, characterized in that: The concentration of the recombinant mussel mucin solution is 5 mg / g-100 mg / g.

10. Use of the gynecological gel containing recombinant mussel mucin according to any one of claims 1 to 7 in the preparation of drugs for preventing or treating gynecological diseases.

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