Slow-release gynecological gel containing recombinant human collagen peptide and preparation method thereof

Abstract

The invention discloses sustained-release gynecological gel containing recombinant human collagen peptide and a preparation method of the sustained-release gynecological gel, and belongs to the field of biological medicines. The preparation method comprises the following steps: stirring and mixing a collagen peptide compound, carbomer, cuba balsam oil and purified water to obtain a mixed emulsion, then adding sodium alginate, and stirring for reaction to obtain the sustained-release gynecological gel containing the recombinant human collagen peptide. According to the sustained-release gynecological gel containing the recombinant human collagen peptide provided by the invention, the occurrence of adhesion is prevented by utilizing the physical isolation of the gel, so that the stability of the collagen and the cuba balsam oil is favorably maintained; the sustained release preparation has the advantages of slow and long-acting release, good stability, high bioavailability, effective reduction of postoperative recurrence rate, good biocompatibility, excellent biodegradability, good tolerance, safety, effectiveness, convenience in use and the like.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to a sustained-release gynecological gel containing recombinant human collagen peptide and a preparation method thereof. Background Art

[0002] The reproductive tract of healthy women has a natural protection function against pathogen invasion, including vaginal pH balance, vaginal orifice closure, and close adhesion of the anterior and posterior vaginal walls, which can maintain the balance among microorganisms in the vagina. However, once stimulated by factors such as endocrine disorders and mucosal damage, the stability of the vaginal environment can be affected, leading to an imbalance in the vaginal environment and inducing various vaginal inflammations. Investigations have found that various gynecological diseases can have an adverse impact on the physical and mental health of patients, and the longer the time, the greater the harm, and even serious diseases such as cervical cancer can be induced. Therefore, continuous attention should be paid in clinical practice, and more scientific and effective treatment methods should be explored to improve the rehabilitation effect of patients and continuously enhance their quality of life. In clinical practice, the main treatment methods for common gynecological diseases are surgery and drug treatment. Among them, drug treatment can be further divided into traditional Chinese medicine treatment, western medicine treatment, and integrated traditional Chinese and western medicine treatment, and various medical technologies have also been continuously developed in clinical practice applications.

[0003] In the prior art, the treatment means for gynecological diseases mainly include three categories: western medicine and antibiotic treatment, physical therapy, and surgical treatment. Among them, western medicine and antibiotic treatment are the most commonly used means, but this method is extremely likely to destroy the flora and acid-base balance of the female reproductive tract, resulting in low immunity and continuous recurrence of inflammation and infection; physical therapy is a relatively healthy treatment means, including electrocautery, laser, etc. Long-term exposure will cause the closure of cervical secretory glands, change cervical mucus, and lead to infertility, and it is also easy to cause cervical dilation disorders during childbirth; surgical treatment is the simplest treatment method and also the most harmful treatment means to the body. Unless in special circumstances, surgical treatment is not recommended. Products related to the maintenance of the female reproductive system in the market are even scarcer.

[0004] Chinese invention patent CN118059215B discloses a gynecological thermosensitive in-situ gel and its preparation and application, which uses thermosensitive gel to encapsulate collagen through hydrophobic interaction. However, collagen has good hydrophilicity and is easy to release rapidly in the initial stage of gel swelling; Chinese invention patent CN114767924B discloses a preparation method of a mussel-inspired adhesive protein hydrogel suitable for the repair of damaged skin cells. Although the hydrogel prolongs the drug release time, it does not introduce mucosal penetration peptides or pH-responsive materials, resulting in insufficient transmembrane transport efficiency of enzymatically hydrolyzed snakehead collagen peptide at the inflammatory site.

[0005] In addition, the sustained-release performance of the gel is not ideal, the drug release rate is too fast or too slow, and it is impossible to achieve precise drug release control, which will also affect the treatment effect and the patient's medication experience. For example, after collagen is hydrolyzed into collagen peptides, its molecular weight decreases, which may lead to a decrease in its retention capacity in the gel system, thereby affecting the sustained-release effect.

[0006] Therefore, providing a gynecological gel with good sustained-release properties and anti-inflammatory properties is an important issue to be solved in the art. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides a sustained-release gynecological gel containing recombinant human collagen peptides and a preparation method thereof. Specifically, the technical solution of the present invention includes the following contents:

[0008] A method for preparing a sustained-release gynecological gel containing recombinant human collagen peptides, the method comprising the following steps:

[0009] The collagen peptide complex, carbomer, copaiba balsam oil and purified water are stirred and mixed to obtain a mixed emulsion, and then sodium alginate is added and stirred for reaction to obtain the sustained-release gynecological gel containing recombinant human collagen peptides.

[0010] Furthermore, the preparation method of the collagen peptide complex comprises the following steps:

[0011] The recombinant human collagen is subjected to a first enzymatic hydrolysis reaction with alkaline protease and a second enzymatic hydrolysis reaction with papain in sequence to obtain a recombinant human collagen peptide; (3-propylcarboxyl) triphenylphosphine bromide, allyloxy polyoxyethylene ether, sodium bisulfate and petroleum ether are subjected to a first mixing reaction to obtain an unsaturated cationic compound; the unsaturated cationic compound and the recombinant human collagen peptide are subjected to a second mixing reaction to obtain a modified recombinant human collagen peptide; the modified recombinant human collagen peptide and a polylysine complex are subjected to a third mixing reaction to obtain the collagen peptide complex.

[0012] Furthermore, the recombinant human collagen is recombinant human type III collagen.

[0013] Furthermore, the preparation method of the polylysine complex comprises the following steps:

[0014] Polylysine and N,N-carbonyldiimidazole are mixed and activated to obtain a mixed solution, the mixed solution and L-cysteine ​​are subjected to a first stirring reaction to obtain modified polylysine, and the modified polylysine and hexahydrophthalic anhydride are subjected to a second stirring reaction to obtain the polylysine complex.

[0015] Furthermore, the reaction pH of the first enzymatic hydrolysis reaction is 8.5-9.5, the reaction temperature is 45-50° C. and the reaction time is 2-3 h.

[0016] Further, the reaction pH of the second enzymatic hydrolysis reaction is 6.0 - 7.0, the reaction temperature is 45 - 50°C, and the reaction time is 2 - 3 h.

[0017] Further, the weight ratio of the (3 - propylcarboxyl) triphenylphosphonium bromide, allyloxy polyoxyethylene ether, sodium bisulfate, and petroleum ether is 3 - 4:0.8 - 1.2:0.3 - 0.4:150 - 300.

[0018] Further, the reaction temperature of the first mixing reaction is 70 - 80°C and the reaction time is 4 - 5 h.

[0019] Further, the weight ratio of the unsaturated bond cationic compound and the recombinant human - derived collagen peptide is 1:0.5 - 0.8.

[0020] Further, the reaction temperature of the second mixing reaction is 30 - 35°C and the reaction time is 48 - 72 h.

[0021] Further, the weight ratio of the modified recombinant human - derived collagen peptide and the polylysine complex is 2 - 3:4 - 5.

[0022] Further, the reaction temperature of the third mixing reaction is 23 - 25°C and the reaction time is 24 - 30 h.

[0023] Further, the weight ratio of the polylysine, N,N - carbonyldiimidazole, and L - cysteine is 1 - 2:0.5 - 1.5:2 - 5.

[0024] Further, the activation temperature of the mixed activation is 30 - 35°C and the activation time is 1 - 2 h.

[0025] Further, the reaction temperature of the first stirring reaction is 23 - 25°C and the reaction time is 24 - 48 h.

[0026] Further, the weight ratio of the modified polylysine and hexahydrophthalic anhydride is 1.5 - 3.5:2 - 4.

[0027] Further, the reaction temperature of the second stirring reaction is 23 - 25°C and the reaction time is 8 - 16 h.

[0028] Further, the carbomer includes one of carbomer 941, carbomer 981, or carbomer 2020.

[0029] Further, the weight ratio of the collagen peptide complex, carbomer, balsam of Peru oil, purified water, and sodium alginate is 2 - 3:4 - 8:1.5 - 2.5:100 - 120:1 - 2.

[0030] Furthermore, the mixing temperature for the stirring and mixing is 35 - 45°C, and the mixing time is 30 - 50 min.

[0031] Furthermore, the reaction temperature for the stirring reaction is 50 - 60°C, and the reaction time is 20 - 30 min.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] (1) In the present invention, recombinant human collagen is treated by a double enzymatic hydrolysis reaction to obtain recombinant human collagen peptides. (3 - propylcarboxyl) triphenylphosphonium bromide and allyloxy polyoxyethylene ether are subjected to an esterification reaction to obtain an unsaturated bond cationic compound. The unsaturated bond cationic compound and the recombinant human collagen peptides undergo a Michael addition reaction to obtain modified recombinant human collagen peptides. Polylysine and L - cysteine are subjected to an amidation reaction to obtain modified polylysine. The modified polylysine reacts with hexahydrophthalic anhydride to obtain a polylysine complex. The modified recombinant human collagen peptides carry a positive charge, and the polylysine complex carries a negative charge. The two are assembled into a collagen peptide complex through electrostatic interaction.

[0034] (2) In the present invention, the collagen peptide complex has pH responsiveness in the slightly acidic environment of inflammatory cells. The structure of the collagen peptide complex becomes unstable, and outer disassembly occurs, realizing the release of the modified recombinant human collagen peptides. The introduction of hydrophobic groups and cationic groups in the modified recombinant human collagen peptides makes the recombinant human collagen peptides bind more closely to the skin surface, increasing their retention amount and action time on the skin surface, exerting their biological activity, and improving the bioavailability. The positively charged recombinant human collagen peptides contribute to their better interaction with lipids and proteins in the skin, enhancing the integrity of the skin barrier. In the present invention, the polylysine complex contains antioxidant groups such as sulfhydryl groups, which can protect balsam copaiba oil from oxidation, thus better exerting the antibacterial and anti - inflammatory effects.

[0035] (3) The sustained - release gynecological gel containing recombinant human collagen peptides of the present invention uses the physical isolation of the gel to prevent adhesion, which is beneficial to maintaining the stability of collagen and balsam copaiba oil. It has the advantages of slow and long - acting release, good stability, high bioavailability, effectively reducing the postoperative recurrence rate, good biocompatibility, excellent biodegradability, good tolerance, safety and effectiveness, and convenient use. Specific Embodiments

[0036] The technical solutions of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0037] Unless otherwise specified, the raw materials and reagents used in the following invention are all commercially available products or can be prepared by known methods.

[0038] Preparation Example 1:

[0039] The preparation of the polylysine complex includes the following steps:

[0040] 1 part by weight of polylysine and 0.5 part by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide and mixed and activated at 30 °C for 1 h to obtain a mixed solution. The obtained mixed solution and 2 parts by weight of L-cysteine are stirred in a nitrogen protection environment at 23 °C for 24 h for the first stirring reaction. After the reaction is completed, it is dialyzed and freeze-dried to obtain modified polylysine; 1.5 parts by weight of the modified polylysine and 2 parts by weight of hexahydrophthalic anhydride are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 = 4:1), the pH is adjusted to 9 and then reacted at 23 °C for 8 h for the second stirring reaction. After the reaction is completed, it is dialyzed in deionized water at pH = 9 and pH = 7.4 for 48 h in sequence to obtain the polylysine complex.

[0041] Preparation Example 2:

[0042] The preparation of the polylysine complex includes the following steps:

[0043] 1.2 parts by weight of polylysine and 0.8 part by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide and mixed and activated at 32 °C for 1.2 h to obtain a mixed solution. The obtained mixed solution and 3 parts by weight of L-cysteine are stirred in a nitrogen protection environment at 24 °C for 30 h for the first stirring reaction. After the reaction is completed, it is dialyzed and freeze-dried to obtain modified polylysine; 2 parts by weight of the modified polylysine and 2.5 parts by weight of hexahydrophthalic anhydride are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 = 4:1), the pH is adjusted to 9.5 and then reacted at 24 °C for 10 h for the second stirring reaction. After the reaction is completed, it is dialyzed in deionized water at pH = 9.5 and pH = 7.4 for 48 h in sequence to obtain the polylysine complex.

[0044] Preparation Example 3:

[0045] The preparation of the polylysine complex includes the following steps:

[0046] 1.6 parts by weight of polylysine and 1.2 parts by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide, and mixed and activated at 32 °C for 1.6 h to obtain a mixed solution. The obtained mixed solution and 4 parts by weight of L-cysteine are stirred at 24 °C for 36 h in a nitrogen protection environment for the first stirring reaction. After the reaction, dialysis and freeze-drying are carried out to obtain modified polylysine; 2.5 parts by weight of modified polylysine and 3 parts by weight of hexahydrophthalic anhydride are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 = 4:1), adjust the pH to 9.8, and react at 24 °C for 14 h for the second stirring reaction. After the reaction, dialysis is carried out in deionized water with pH = 9.5 and pH = 7.4 for 48 h in sequence to obtain a polylysine complex.

[0047] Preparation Example 4:

[0048] Preparation of polylysine complex, including the following steps:

[0049] 2 parts by weight of polylysine and 1.5 parts by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide, and mixed and activated at 35 °C for 2 h to obtain a mixed solution. The obtained mixed solution and 5 parts by weight of L-cysteine are stirred at 25 °C for 48 h in a nitrogen protection environment for the first stirring reaction. After the reaction, dialysis and freeze-drying are carried out to obtain modified polylysine; 3.5 parts by weight of modified polylysine and 4 parts by weight of hexahydrophthalic anhydride are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 = 4:1), adjust the pH to 10, and react at 25 °C for 16 h for the second stirring reaction. After the reaction, dialysis is carried out in deionized water with pH = 10 and pH = 7.4 for 48 h in sequence to obtain a polylysine complex.

[0050] Preparation Example 5:

[0051] Preparation of polylysine complex, including the following steps:

[0052] 2 parts by weight of polylysine and 4 parts by weight of hexahydrophthalic anhydride are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 = 4:1), adjust the pH to 10, and react at 25 °C for 16 h for the second stirring reaction. After the reaction, dialysis is carried out in deionized water with pH = 10 and pH = 7.4 for 48 h in sequence to obtain a polylysine complex.

[0053] Preparation Example 6:

[0054] Preparation of polylysine complex, including the following steps:

[0055] 2 parts by weight of polylysine and 1.5 parts by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide and mixed and activated at 35°C for 2 h to obtain a mixed solution. The obtained mixed solution and 5 parts by weight of L-cysteine are stirred at 25°C for 48 h in a nitrogen protection environment for the first stirring reaction. After the reaction is completed, it is dialyzed and freeze-dried to obtain modified polylysine; 3.5 parts by weight of the modified polylysine and 4 parts by weight of phthalic acid are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 =4:1), adjust the pH to 10 and react at 25°C for 16 h for the second stirring reaction. After the reaction is completed, it is dialyzed in deionized water with pH = 10 and pH = 7.4 for 48 h in sequence to obtain a polylysine complex.

[0056] Preparation Example 7:

[0057] Preparation of polylysine complex, including the following steps:

[0058] 2 parts by weight of polyethyleneimine and 1.5 parts by weight of N,N'-carbonyldiimidazole are dispersed in 100 parts by weight of dimethyl sulfoxide and mixed and activated at 35°C for 2 h to obtain a mixed solution. The obtained mixed solution and 5 parts by weight of L-cysteine are stirred at 25°C for 48 h in a nitrogen protection environment for the first stirring reaction. After the reaction is completed, it is dialyzed and freeze-dried to obtain modified polylysine; 3.5 parts by weight of the modified polylysine and 4 parts by weight of phthalic acid are dispersed in 300 parts by weight of a mixed solvent (V 去离子水 :V 乙醇 =4:1), adjust the pH to 10 and react at 25°C for 16 h for the second stirring reaction. After the reaction is completed, it is dialyzed in deionized water with pH = 10 and pH = 7.4 for 48 h in sequence to obtain a polylysine complex.

[0059] Preparation Example 8:

[0060] Preparation of collagen peptide complex, including the following steps:

[0061] 2 parts by weight of recombinant human type III collagen are dispersed in 40 parts by weight of deionized water. After being kept at 50 °C for 10 min, 0.04 part by weight of alkaline protease is added and the pH is adjusted to 8.5. The first enzymatic hydrolysis reaction is carried out at 45 °C for 2 h. After the reaction is completed, it is treated at 95 °C for 10 min to inactivate the enzyme. After cooling to 23 °C, it is centrifuged at a speed of 4000 r / min for 20 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.04 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 6.0. Then the second enzymatic hydrolysis reaction is carried out at 45 °C for 2 h. After the reaction is completed, it is treated at 95 °C for 10 min to inactivate the enzyme. After cooling to 23 °C, it is centrifuged at a speed of 4000 r / min for 20 min to collect the supernatant to obtain recombinant human collagen peptide; 3 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 0.8 part by weight of allyloxy polyoxyethylene ether are dispersed in 150 parts by weight of petroleum ether. Then 0.3 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 70 °C for 4 h. After the reaction is completed, it is obtained by rotary evaporation, washing and recrystallization to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.5 part by weight of the recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 30 °C for 48 h. After the reaction is completed, it is purified by extraction, ion exchange chromatography and freeze-drying steps in sequence to obtain the modified collagen peptide; 2 parts by weight of the modified recombinant human collagen peptide and 4 parts by weight of the polylysine complex prepared in Preparation Example 1 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 23 °C for 24 h. After the reaction is completed, it is dialyzed and freeze-dried to obtain the collagen peptide complex.

[0062] Preparation Example 9:

[0063] Preparation of the collagen peptide complex, comprising the following steps:

[0064] 2.3 parts by weight of recombinant human type III collagen are dispersed in 45 parts by weight of deionized water. After being kept warm at 55 °C for 12 min, 0.045 part by weight of alkaline protease is added and the pH is adjusted to 9. The first enzymatic hydrolysis reaction is carried out at 46 °C for 2.2 h. After the reaction ends, it is treated at 97 °C for 12 min to inactivate the enzyme. After cooling to 24 °C, it is centrifuged at a speed of 4000 r / min for 24 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.043 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 6.3. Then the second enzymatic hydrolysis reaction is carried out at 47 °C for 2.1 h. After the reaction ends, it is treated at 98 °C for 12 min to inactivate the enzyme. After cooling to 24 °C, it is centrifuged at a speed of 4000 r / min for 23 min to collect the supernatant to obtain recombinant human collagen peptide; 3.2 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 0.9 part by weight of allyloxy polyoxyethylene ether are dispersed in 180 parts by weight of petroleum ether. Then 0.32 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 73 °C for 4.3 h. After the reaction ends, it is obtained by rotary evaporation, washing and recrystallization to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.7 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 32 °C for 54 h. After the reaction ends, it is purified by steps such as extraction, ion exchange chromatography and freeze-drying to obtain modified collagen peptide; 2.5 parts by weight of modified recombinant human collagen peptide and 4.3 parts by weight of the polylysine complex prepared in Preparation Example 2 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 24 °C for 26 h. After the reaction ends, it is obtained by dialysis and freeze-drying to prepare a collagen peptide complex.

[0065] Preparation Example 10:

[0066] Preparation of the collagen peptide complex, comprising the following steps:

[0067] 2.6 parts by weight of recombinant human type III collagen are dispersed in 50 parts by weight of deionized water. After incubating at 56 °C for 13 min, 0.052 part by weight of alkaline protease is added and the pH is adjusted to 9.1. The first enzymatic hydrolysis reaction is carried out at 48 °C for 2.6 h. After the reaction is completed, it is treated at 98 °C for 12 min to inactivate the enzyme. After cooling to 24 °C, it is centrifuged at 4000 r / min for 26 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.05 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 6.7. Then, the second enzymatic hydrolysis reaction is carried out at 48 °C for 2.8 h. After the reaction is completed, it is treated at 98 °C for 12 min to inactivate the enzyme. After cooling to 24 °C, it is centrifuged at 4000 r / min for 28 min to collect the supernatant to obtain recombinant human collagen peptide; 3.5 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1 part by weight of allyloxy polyoxyethylene ether are dispersed in 250 parts by weight of petroleum ether. Then, 0.38 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 78 °C for 4.8 h. After the reaction is completed, it is obtained by rotary evaporation, washing and recrystallization to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.7 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 34 °C for 64 h. After the reaction is completed, it is purified by extraction, ion exchange chromatography, freeze-drying and other steps to obtain modified collagen peptide; 2.8 parts by weight of modified recombinant human collagen peptide and 4.6 parts by weight of the polylysine complex prepared in Preparation Example 3 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 24 °C for 28 h. After the reaction is completed, it is dialyzed and freeze-dried to obtain a collagen peptide complex.

[0068] Preparation Example 11:

[0069] Preparation of the collagen peptide complex, comprising the following steps:

[0070] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubation at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treatment at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product; 0.06 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0. Then, the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treatment at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether. Then, 0.4 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 80 °C for 5 h. After the reaction is completed, the unsaturated bond cationic compound is obtained by rotary evaporation, washing and recrystallization; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of the recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 35 °C for 72 h. After the reaction is completed, the modified collagen peptide is obtained by purification through steps such as extraction, ion exchange chromatography and freeze-drying; 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Preparation Example 4 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 25 °C for 30 h. After the reaction is completed, the collagen peptide complex is obtained by dialysis and freeze-drying.

[0071] Preparation Example 12:

[0072] Preparation of the collagen peptide complex, comprising the following steps:

[0073] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubation at 60 °C for 15 min, 0.06 parts by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product; 0.06 parts by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0. Then, the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether. Then, 0.4 parts by weight of sodium bisulfate is added and the first mixing reaction is carried out at 80 °C for 5 h. After the reaction is completed, the unsaturated bond cationic compound is obtained by rotary evaporation, washing and recrystallization; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 35 °C for 72 h. After the reaction is completed, the modified collagen peptide is obtained by purification through steps such as extraction, ion exchange chromatography and freeze-drying; 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Preparation Example 5 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 25 °C for 30 h. After the reaction is completed, the collagen peptide complex is obtained by dialysis and freeze-drying.

[0074] Preparation Example 13:

[0075] Preparation of the collagen peptide complex, comprising the following steps:

[0076] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubation at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product; 0.06 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0. Then, the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, the enzyme is inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant is collected by centrifugation at 4000 r / min for 30 min to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether. Then, 0.4 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 80 °C for 5 h. After the reaction is completed, the unsaturated bond cationic compound is obtained by rotary evaporation, washing and recrystallization; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of the recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) mixed solvent. The second mixing reaction is carried out at 35 °C for 72 h. After the reaction is completed, purification is carried out through steps such as extraction, ion exchange chromatography and freeze-drying to obtain the modified collagen peptide; 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Preparation Example 6 are dispersed in 200 parts by weight of deionized water. The third mixing reaction is carried out at 25 °C for 30 h. After the reaction is completed, dialysis and freeze-drying are carried out to obtain the collagen peptide complex.

[0077] Preparation Example 14:

[0078] Preparation of the collagen peptide complex, comprising the following steps:

[0079] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubation at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.06 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0. Then the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether, and then 0.4 part by weight of sodium bisulfate is added and the reaction is carried out at 80 °C for 5 h for the first mixing reaction. After the reaction is completed, it is obtained by rotary evaporation, washing and recrystallization to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 =4:1) mixed solvent, and the second mixing reaction is carried out at 35 °C for 72 h. After the reaction is completed, it is purified by extraction, ion exchange chromatography, freeze-drying and other steps to obtain modified collagen peptide; 3 parts by weight of modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Preparation Example 7 are dispersed in 200 parts by weight of deionized water, and the third mixing reaction is carried out at 25 °C for 30 h. After the reaction is completed, it is obtained by dialysis and freeze-drying to obtain a collagen peptide complex.

[0080] Preparation Example 15:

[0081] Preparation of collagen peptide complex, comprising the following steps:

[0082] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubation at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether, and then 0.4 part by weight of sodium bisulfate is added and the reaction is carried out at 80 °C for 5 h for the first mixing reaction. After the reaction is completed, it is obtained by rotary evaporation, washing and recrystallization to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :VN,N-二甲基甲酰胺 = 4:1) In the mixed solvent, the second mixing reaction was carried out at 35 °C for 72 h. After the reaction, purification was carried out through steps such as extraction, ion exchange chromatography, and freeze-drying to obtain modified collagen peptides; 3 parts by weight of modified recombinant human collagen peptides and 5 parts by weight of the polylysine complex prepared in Preparation Example 4 were dispersed in 200 parts by weight of deionized water, and the third mixing reaction was carried out at 25 °C for 30 h. After the reaction, dialysis and freeze-drying were carried out to obtain the collagen peptide complex.

[0083] Preparation Example 16:

[0084] The preparation of the collagen peptide complex includes the following steps:

[0085] 3 parts by weight of recombinant human type III collagen was dispersed in 60 parts by weight of deionized water. After maintaining the temperature at 60 °C for 15 min, 0.06 parts by weight of papain was added and the pH was adjusted to 7.0. Then, an enzymatic hydrolysis reaction was carried out at 50 °C for 3 h. After the reaction, the enzyme was inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant was collected by centrifuging at a speed of 4000 r / min for 30 min to obtain recombinant human collagen peptides; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether were dispersed in 300 parts by weight of petroleum ether, and then 0.4 parts by weight of sodium bisulfate was added and the first mixing reaction was carried out at 80 °C for 5 h. After the reaction, rotary evaporation, washing, and recrystallization were carried out to obtain an unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of recombinant human collagen peptides were dispersed in 50 parts by weight (V 去离子水 :V N,N-二甲基甲酰胺 = 4:1) In the mixed solvent, the second mixing reaction was carried out at 35 °C for 72 h. After the reaction, purification was carried out through steps such as extraction, ion exchange chromatography, and freeze-drying to obtain modified collagen peptides; 3 parts by weight of modified recombinant human collagen peptides and 5 parts by weight of the polylysine complex prepared in Preparation Example 4 were dispersed in 200 parts by weight of deionized water, and the third mixing reaction was carried out at 25 °C for 30 h. After the reaction, dialysis and freeze-drying were carried out to obtain the collagen peptide complex.

[0086] Preparation Example 17:

[0087] The preparation of the collagen peptide complex includes the following steps:

[0088] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubating at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.06 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0, then the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide, 2.5 parts by weight of 1,3-dicyclohexylcarbodiimide and 2 parts by weight of N-hydroxysuccinimide are dispersed in 100 parts by weight of N,N-dimethylformamide and stirred at 60 °C for 4 h, then 0.8 part by weight of recombinant human collagen peptide is added and stirred at 23 °C for 24 h. After the reaction is completed, it is rotary evaporated, dialyzed and dried to obtain the modified collagen peptide; 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Preparation Example 4 are dispersed in 200 parts by weight of deionized water and reacted at 25 °C for 30 h for the third mixing reaction. After the reaction is completed, it is dialyzed and freeze-dried to obtain the collagen peptide complex.

[0089] Preparation Example 18:

[0090] Preparation of the modified collagen peptide, comprising the following steps:

[0091] 3 parts by weight of recombinant human type III collagen are dispersed in 60 parts by weight of deionized water. After incubating at 60 °C for 15 min, 0.06 part by weight of alkaline protease is added and the pH is adjusted to 9.5. The first enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain the enzymatic hydrolysis product; 0.06 part by weight of papain is added to 100 parts by weight of the enzymatic hydrolysis product and the pH is adjusted to 7.0, then the second enzymatic hydrolysis reaction is carried out at 50 °C for 3 h. After the reaction is completed, it is treated at 100 °C for 15 min to inactivate the enzyme. After cooling to 25 °C, it is centrifuged at a speed of 4000 r / min for 30 min to collect the supernatant to obtain recombinant human collagen peptide; 4 parts by weight of (3-carboxypropyl) triphenylphosphonium bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether are dispersed in 300 parts by weight of petroleum ether, then 0.4 part by weight of sodium bisulfate is added and the first mixing reaction is carried out at 80 °C for 5 h. After the reaction is completed, it is rotary evaporated, washed and recrystallized to obtain the unsaturated bond cationic compound; 1 part by weight of the unsaturated bond cationic compound and 0.8 part by weight of recombinant human collagen peptide are dispersed in 50 parts by weight (V 去离子水 :VN,N-二甲基甲酰胺 In a mixed solvent with a volume ratio of 4:1, the second mixing reaction was carried out at 35 °C for 72 h. After the reaction, purification was carried out through steps such as extraction, ion exchange chromatography, and freeze-drying to obtain modified collagen peptides.

[0092] Preparation Example 19:

[0093] The preparation of collagen peptides includes the following steps:

[0094] 3 parts by weight of recombinant human type III collagen was dispersed in 60 parts by weight of deionized water. After maintaining the temperature at 60 °C for 15 min, 0.06 parts by weight of alkaline protease was added and the pH was adjusted to 9.5. The first enzymatic hydrolysis reaction was carried out at 50 °C for 3 h. After the reaction, the enzyme was inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant was collected by centrifuging at a speed of 4000 r / min for 30 min to obtain the enzymatic hydrolysis product; 0.06 parts by weight of papain was added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. Then, the second enzymatic hydrolysis reaction was carried out at 50 °C for 3 h. After the reaction, the enzyme was inactivated by treating at 100 °C for 15 min. After cooling to 25 °C, the supernatant was collected by centrifuging at a speed of 4000 r / min for 30 min to obtain recombinant human-derived collagen peptides.

[0095] Example 1:

[0096] The preparation of a sustained-release gynecological gel containing recombinant human-derived collagen peptides includes the following steps:

[0097] 2 parts by weight of the collagen peptide complex prepared in Preparation Example 8, 4 parts by weight of carbomer 491, and 1.5 parts by weight of balsam of Peru were dispersed in 100 parts by weight of purified water. The mixture was stirred and mixed at 35 °C for 30 min to obtain a mixed emulsion. Then, 1 part by weight of sodium alginate was added to the mixed emulsion, and the temperature was raised to 50 °C and stirred for 30 min to obtain a sustained-release gynecological gel containing recombinant human-derived collagen peptides.

[0098] Example 2:

[0099] The preparation of a sustained-release gynecological gel containing recombinant human-derived collagen peptides includes the following steps:

[0100] 2.3 parts by weight of the collagen peptide complex prepared in Preparation Example 9, 5 parts by weight of carbomer 981, and 1.8 parts by weight of balsam of Peru were dispersed in 110 parts by weight of purified water. The mixture was stirred and mixed at 38 °C for 35 min to obtain a mixed emulsion. Then, 1.4 parts by weight of sodium alginate was added to the mixed emulsion, and the temperature was raised to 52 °C and stirred for 28 min to obtain a sustained-release gynecological gel containing recombinant human-derived collagen peptides.

[0101] Example 3:

[0102] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0103] 2.6 parts by weight of the collagen peptide complex prepared in Preparation Example 10, 6.5 parts by weight of carbomer 2020 and 2.1 parts by weight of balsam of Peru are dispersed in 115 parts by weight of purified water, stirred and mixed at 42 °C for 45 min to obtain a mixed emulsion, and then 1.6 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 55 °C and stirred and reacted for 25 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0104] Example 4:

[0105] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0106] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 11, 8 parts by weight of carbomer 981 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0107] Comparative Example 1:

[0108] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0109] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 12, 8 parts by weight of carbomer 981 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0110] Comparative Example 2:

[0111] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0112] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 13, 8 parts by weight of carbomer 981 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0113] Comparative Example 3:

[0114] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0115] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 14, 8 parts by weight of carbomer 981, and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0116] Comparative Example 4:

[0117] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0118] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 15, 8 parts by weight of carbomer 981, and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0119] Comparative Example 5:

[0120] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0121] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 16, 8 parts by weight of carbomer 981, and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0122] Comparative Example 6:

[0123] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0124] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 17, 8 parts by weight of carbomer 981, and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0125] Comparative Example 7:

[0126] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0127] 3 parts by weight of the modified collagen peptide prepared in Preparation Example 18, 8 parts by weight of carbomer 981 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0128] Comparative Example 8:

[0129] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0130] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 12 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0131] Comparative Example 9:

[0132] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0133] 3 parts by weight of the collagen peptide complex prepared in Preparation Example 12 and 8 parts by weight of carbomer 981 are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0134] Comparative Example 10:

[0135] Preparation of a sustained-release gynecological gel containing recombinant human collagen peptide, comprising the following steps:

[0136] 3 parts by weight of the collagen peptide prepared in Preparation Example 19, 8 parts by weight of carbomer 981 and 2.5 parts by weight of balsam of Peru are dispersed in 120 parts by weight of purified water, stirred and mixed at 45 °C for 50 min to obtain a mixed emulsion, and then 2 parts by weight of sodium alginate is added to the mixed emulsion, and the temperature is raised to 60 °C and stirred and reacted for 20 min to prepare a sustained-release gynecological gel containing recombinant human collagen peptide.

[0137] Performance test:

[0138] Performance tests were conducted on the sustained-release gynecological gels containing recombinant human-derived collagen peptides prepared in Examples 1-4 and Comparative Examples 1-10 of the present invention, and the test results are as follows.

[0139] Test Example 1: Irritation test

[0140] Forty-five healthy female rabbits were randomly divided into 15 groups. Groups 1-15 corresponded to Examples 1-4, Comparative Examples 1-10, and the blank group, respectively. Then, 2 mL of the sustained-release gynecological gel containing recombinant human-derived collagen peptides prepared in Examples 1-4 and Comparative Examples 1-10 was injected into Groups 1-14 every day for 7 consecutive days. The blank group was not treated. The vagina of the rabbits was observed every day for redness or erosion. Scoring was based on the irritation degree judgment criteria: no redness - 0 points, slightly red - 1 point, severely red - 2 points, slightly eroded - 3 points, severely eroded - 4 points. The scores for each day were summarized and statistically analyzed. The statistical results are shown in Table 1 below.

[0141] Table 1. Results of the irritation test

[0142]

[0143]

[0144] The experimental results in Table 1 show that the sustained-release gynecological gel containing recombinant human-derived collagen peptides prepared in the present invention has less irritation and good safety.

[0145] Test Example 2: Animal experiment

[0146] Healthy female rats were selected for the establishment of a comprehensive vaginitis model. On the third day before applying the experimental sample gel, estradiol benzoate injection was subcutaneously injected into the rats every morning, with a dosage of 0.5 mg / rat / time. Streptomycin was also injected into the vagina of the rats at a dosage of 50 μg / rat / time. Then, a mixed bacterium with a concentration of 1×108 CFU / mL, composed of Gardnerella vaginalis, Candida albicans, and Group B streptococcus with a colony number ratio of 1:1:1, was injected into the vagina at a dosage of 50 μg / rat / time. All injection substances were administered once a day for 3 consecutive days. The rats in the blank group were inoculated with 50 μL of PBS without strains into the vagina. After 5 days, the vaginas of the rats were observed. If the vulva and vagina of the rats showed swelling, thickening, and the secretions became cheesy or curd-like, the model establishment was considered successful. 48 successfully modeled rats were randomly and evenly divided into 16 groups, with 3 rats in each group. Groups 1-4 corresponded to Examples 1-4, groups 5-14 corresponded to Comparative Examples 1-10, group 15 corresponded to the positive control group, and group 16 was the negative control group. The rats in group 16 were healthy rats without comprehensive vaginitis model establishment treatment. Groups 1-14 were respectively smeared with the sustained-release gynecological gel containing recombinant human collagen peptide prepared in Examples 1-4 and Comparative Examples 1-10, at a dosage of 0.5 g / kg body weight once a day for 7 consecutive days. The positive control group in group 15 was smeared with the same amount of sterile normal saline once a day for 7 consecutive days. After 7 days, the rats in Examples 1-4, Comparative Examples 1-10, the positive control group, and the negative control group were sacrificed respectively. Then, the vaginal tissues of the rats were homogenized, centrifuged at 4000 r / min for 10 min, and the precipitate was discarded. The contents of IL-6 and IL-8 in the vaginal tissues of the rats in Examples 1-4, Comparative Examples 1-10, the positive control group, and the negative control group were detected using an ELISA kit, and the reduction rates (%) of IL-6 and IL-8 in Examples 1-4 and Comparative Examples 1-10 were calculated. The experimental results were averaged, and the results are shown in Table 2.

[0147] Reduction rate (%) = (M1 - M0) / M0 × 100%, where M1 is the content of IL-6 or IL-8 in Examples 1-4, Comparative Examples 1-10, and the positive control group; M0 is the content of IL-6 or IL-8 in the negative control group;

[0148] Table 2. Results of animal experiments

[0149]

[0150]

[0151] From the experimental results in Table 2, it can be observed that the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Examples 1 to 4 of the present invention can effectively reduce the content of inflammatory factors IL-6 or IL-8 in the vaginal tissues of rats, have a good antibacterial and anti-inflammatory effect, have a repair effect on damaged vaginas, and effectively promote the treatment effect of gynecological diseases such as vaginitis.

[0152] Test Example 3: Detection of in vitro release effect

[0153] Weigh 10 mg of the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Examples 1 to 4 and Comparative Examples 1, 2, 3, 7, and 10, respectively, add them to 50 mL centrifuge tubes, immerse them in 30 mL of PBS solutions with pH = 3.5, pH = 5.0, and pH = 7.0, respectively, then place them in a constant temperature oscillator at 100 rpm and 37 °C. At different times, take out 3 nL of the supernatant solution and replace it with an equal amount of fresh PBS solution, and use ultraviolet-visible spectrophotometry to measure the concentration of recombinant human collagen peptides. The detection results are shown in Tables 3, 4, and 5.

[0154] Table 3. Detection of in vitro release effect (pH = 3.5)

[0155]

[0156]

[0157] Table 4. Detection of in vitro release effect (pH = 5.0)

[0158]

[0159]

[0160] Table 5. Detection of in vitro release effect (pH = 7.4)

[0161]

[0162] From the experimental results in Tables 3, 4, and 5, it can be obtained that the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Examples 1 to 4 of the present invention have good responsiveness and sustained-release performance in the inflammatory microenvironment, the sustained-release effect is stable, which is beneficial to repairing the damaged skin barrier, has a longer nursing period, improves the repair effect of damaged cells, and will rapidly release modified collagen peptides in the normal vaginal acidic environment; it shows a responsive sustained-release effect in the slightly acidic environment of vaginal inflammation. The sustained-release effects of the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Comparative Examples 1, 2, 3, 7, and 10 are not as good as those in Examples 1 to 4, and it is difficult to achieve long-term treatment.

[0163] The above-described embodiments have detailed the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide, characterized in that, The preparation method comprises the following steps: After stirring and mixing collagen peptide complex, carbomer, balsam of Peru oil and purified water to obtain a mixed emulsion, sodium alginate is added and stirred to react to obtain the sustained-release gynecological gel containing recombinant human collagen peptide.

2. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 1, characterized in that, The preparation method of the collagen peptide complex comprises the following steps: Recombinant human collagen is successively subjected to a first enzymatic hydrolysis reaction with alkaline protease and a second enzymatic hydrolysis reaction with papain to obtain recombinant human collagen peptide; (3-propylcarboxyl) triphenylphosphonium bromide, allyloxy polyoxyethylene ether, sodium bisulfate and petroleum ether are subjected to a first mixing reaction to obtain an unsaturated bond cationic compound; the unsaturated bond cationic compound and recombinant human collagen peptide are subjected to a second mixing reaction to obtain a modified recombinant human collagen peptide; the modified recombinant human collagen peptide and polylysine complex are subjected to a third mixing reaction to obtain the collagen peptide complex.

3. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 2, wherein The preparation method of the polylysine complex comprises the following steps: Polylysine and N,N'-carbonyldiimidazole are mixed and activated to obtain a mixed solution, the mixed solution and L-cysteine are subjected to a first stirring reaction to obtain modified polylysine, and the modified polylysine and hexahydrophthalic anhydride are subjected to a second stirring reaction to obtain the polylysine complex.

4. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 2, characterized in that, The weight ratio of (3-propylcarboxyl) triphenylphosphonium bromide, allyloxy polyoxyethylene ether, sodium bisulfate and petroleum ether is 3-4: 0.8～1.2：0.3～0.4：150～300。 5. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide as described in claim 2, characterized in that, The weight ratio of the unsaturated bond cationic compound and recombinant human collagen peptide is 1:0.5-0.

8.

6. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide as described in claim 2, characterized in that, The weight ratio of the modified recombinant human collagen peptide and polylysine complex is 2-3:4-5.

7. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 3, characterized in that, The weight ratio of polylysine, N,N'-carbonyldiimidazole and L-cysteine is 1-2: 0.5～1.5：2～5。 8. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 3, wherein The weight ratio of the modified polylysine and hexahydrophthalic anhydride is 1.5-3.5:2-4.

9. The preparation method of a sustained-release gynecological gel containing recombinant human collagen peptide according to claim 1, characterized in that, The weight ratio of the collagen peptide complex, carbomer, balsam of Peru oil, purified water and sodium alginate is 2-3:4-8:1.5-2.5:100-120:1-2.

10. A sustained-release gynecological gel containing recombinant human collagen peptide, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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