A sustained-release gynecological gel containing a recombinant human collagen peptide and a preparation method thereof

Abstract

The application discloses a sustained-release gynecological gel containing recombinant human collagen peptide and a preparation method thereof, and belongs to the field of biological medicines. The preparation method comprises the following steps: stirring and mixing collagen peptide compound, carbomer, copaiba balsam oil and purified water to obtain a mixed emulsion, and then adding sodium alginate to stir and react to prepare the sustained-release gynecological gel containing the recombinant human collagen peptide. The sustained-release gynecological gel containing the recombinant human collagen peptide has the advantages of preventing the occurrence of adhesion by physical isolation of the gel, being beneficial to maintaining the stability of collagen and copaiba balsam oil, slowly and long-acting releasing, good stability, high bioavailability, effectively reducing the postoperative recurrence rate, good biocompatibility, excellent biodegradability, good tolerance, safety and effectiveness, and convenient use.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a sustained-release gynecological gel containing recombinant human collagen peptides and its preparation method. Background Technology

[0002] A healthy female reproductive tract has natural protective functions against pathogen invasion, including maintaining vaginal pH balance, vaginal opening closure, and tight apposition of the anterior and posterior vaginal walls, thus preserving the balance of vaginal microorganisms. However, stimulation from factors such as endocrine disorders and mucosal damage can affect the stability of the vaginal environment, leading to imbalance and various vaginal inflammations. Studies have found that various gynecological diseases can adversely affect the physical and mental health of patients, and the longer the disease persists, the greater the harm, even potentially inducing serious diseases such as cervical cancer. Therefore, it is crucial to pay close attention to these issues in clinical practice, continuously exploring more scientific and effective treatment methods to improve patient recovery and quality of life. In clinical practice, common gynecological diseases are mainly treated with surgery and medication. Medication treatment can be further subdivided into traditional Chinese medicine, Western medicine, and integrated traditional Chinese and Western medicine treatments. Various medical technologies are also continuously developing and being applied in clinical practice.

[0003] Currently, treatments for gynecological diseases mainly fall into three categories: Western medicine and antibiotics, physical therapy, and surgery. Western medicine and antibiotics are the most commonly used methods, but these methods easily disrupt the flora and pH balance of the female reproductive tract, leading to weakened immunity and persistent, recurring infections. Physical therapy, including electrocautery and laser treatments, is a relatively healthy approach; however, long-term exposure can cause cervical gland blockage, altering cervical mucus and potentially leading to infertility, and can also cause cervical dilation problems during childbirth. Surgery is the simplest treatment method but also the most harmful to the body; it is generally not recommended unless there are special circumstances. Furthermore, products related to the care of the female reproductive system are scarce in the market.

[0004] Chinese invention patent CN118059215B discloses a gynecological thermosensitive in-situ gel and its preparation and application. The thermosensitive gel relies on hydrophobic interactions to embed collagen. However, collagen has good hydrophilicity and is easily released rapidly in the early stage of gel swelling. Chinese invention patent CN114767924B discloses a method for preparing a mussel-like adhesive protein hydrogel suitable for repairing damaged skin cells. Although the hydrogel prolongs the drug release time, it does not introduce mucosal penetrating peptides or pH-responsive materials, resulting in insufficient transmembrane transport efficiency of enzymatically hydrolyzed snakehead collagen peptides at the site of inflammation.

[0005] Furthermore, if the sustained-release properties of the gel are not ideal, with drug release rates that are too fast or too slow, precise drug release control cannot be achieved, which can also affect the therapeutic effect and the patient's medication experience. For example, after collagen is hydrolyzed into collagen peptides, its molecular weight decreases, which may reduce its retention capacity in the gel system, thereby affecting the sustained-release effect.

[0006] Therefore, providing a gynecological gel with good sustained-release and anti-inflammatory properties is an important problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a sustained-release gynecological gel containing recombinant human collagen peptides and its preparation method. Specifically, the technical solution of this invention includes the following:

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

[0009] Collagen peptide complex, carbomer, Cuban balsam oil and purified water were mixed to obtain a mixed emulsion, and then sodium alginate was added and stirred to obtain the sustained-release gynecological gel containing recombinant human collagen peptides.

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

[0011] Recombinant human collagen was subjected to a first enzymatic hydrolysis reaction by alkaline protease and a second enzymatic hydrolysis reaction by papain to obtain recombinant human collagen peptides; (3-propanoyl)triphenylphosphine bromide, allyloxy polyoxyethylene ether, sodium bisulfate and petroleum ether were subjected to a first mixed reaction to obtain an unsaturated cationic compound; the unsaturated cationic compound and the recombinant human collagen peptides were subjected to a second mixed reaction to obtain modified recombinant human collagen peptides; the modified recombinant human collagen peptides and polylysine complexes were subjected to a third mixed 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 includes the following steps:

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

[0015] Furthermore, the first enzymatic hydrolysis reaction has a reaction pH of 8.5-9.5, a reaction temperature of 45-50℃, and a reaction time of 2-3 hours.

[0016] Furthermore, the second enzymatic hydrolysis reaction is carried out at a pH of 6.0-7.0, a temperature of 45-50°C, and a reaction time of 2-3 hours.

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

[0018] Furthermore, the reaction temperature of the first mixed reaction is 70~80℃ and the reaction time is 4~5h.

[0019] Furthermore, the weight ratio of the unsaturated cationic compound to the recombinant human collagen peptide is 1:0.5~0.8.

[0020] Furthermore, the reaction temperature of the second mixed reaction is 30~35℃ and the reaction time is 48~72h.

[0021] Furthermore, the weight ratio of the modified recombinant human collagen peptide to the polylysine complex is 2~3:4~5.

[0022] Furthermore, the reaction temperature of the third mixed reaction is 23~25℃ and the reaction time is 24~30h.

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

[0024] Furthermore, the activation temperature for the mixed activation is 30~35℃ and the activation time is 1~2h.

[0025] Furthermore, the reaction temperature of the first stirring reaction is 23~25℃ and the reaction time is 24~48h.

[0026] Furthermore, the weight ratio of the modified polylysine to hexahydrophthalic anhydride is 1.5~3.5:2~4.

[0027] Furthermore, the reaction temperature of the second stirring reaction is 23~25℃ and the reaction time is 8~16h.

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

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

[0030] Furthermore, the mixing temperature is 35~45℃ and the mixing time is 30~50min.

[0031] Furthermore, the reaction temperature of the stirring reaction is 50~60℃ and the reaction time is 20~30min.

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

[0033] (1) In this invention, recombinant human collagen peptides are obtained by treating recombinant human collagen with a double enzymatic hydrolysis reaction. (3-propanoyl)triphenylphosphine bromide and allyloxy polyoxyethylene ether are esterified to obtain an unsaturated cationic compound. The unsaturated cationic compound and the recombinant human collagen peptide are reacted with Michael addition reaction to obtain modified recombinant human collagen peptides. Polylysine and L-cysteine ​​are amidated to obtain modified polylysine. Modified polylysine is reacted with hexahydrophthalic anhydride to obtain polylysine complex. The modified recombinant human collagen peptides are positively charged and the polylysine complex is negatively charged. The two are assembled into collagen peptide complex through electrostatic interaction.

[0034] (2) In this invention, the collagen peptide complex exhibits pH responsiveness in the slightly acidic environment of inflammatory cells, causing the collagen peptide complex structure to become unstable and undergo outer layer disassembly, thereby releasing the modified recombinant human collagen peptide. The introduction of hydrophobic and cationic groups into the modified recombinant human collagen peptide allows it to bind more tightly to the skin surface, increasing its retention and duration of action on the skin surface, thus enhancing its bioactivity and bioavailability. The positively charged recombinant human collagen peptide helps it interact better with lipids and proteins in the skin, enhancing the integrity of the skin barrier. The polylysine complex in this invention contains antioxidant groups such as thiol groups, which can protect the Cuban balsam oil from oxidation, thereby better exerting its antibacterial and anti-inflammatory effects.

[0035] (3) The sustained-release gynecological gel containing recombinant human collagen peptides of the present invention utilizes the physical isolation of the gel to prevent adhesion, which helps to maintain the stability of collagen and Cuban balsam oil. It has the advantages of slow and long-lasting release, good stability, high bioavailability, effective reduction of postoperative recurrence rate, good biocompatibility, excellent biodegradability, good tolerance, safety and effectiveness, and convenient use. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below through embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

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

[0038] Preparation Example 1:

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

[0040] One part by weight of polylysine and 0.5 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 30°C for 1 hour to obtain a mixture. The mixture obtained above and 2 parts by weight of L-cysteine ​​were stirred at 23°C for 24 hours under nitrogen protection to carry out the first stirring reaction. After the reaction was completed, modified polylysine was obtained by dialysis and freeze-drying. 1.5 parts by weight of modified polylysine and 2 parts by weight of hexahydrophthalic anhydride were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of 4:1 was adjusted to pH 9, and the reaction was carried out at 23°C for 8 hours for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 9 and pH 7.4 for 48 hours in sequence.

[0041] Preparation Example 2:

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

[0043] 1.2 parts by weight of polylysine and 0.8 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 32°C for 1.2 h to obtain a mixture. The mixture obtained above and 3 parts by weight of L-cysteine ​​were stirred at 24°C for 30 h under nitrogen protection for the first stirring reaction. After the reaction was completed, modified polylysine was obtained by dialysis and freeze-drying. 2 parts by weight of modified polylysine and 2.5 parts by weight of hexahydrophthalic anhydride were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of 4:1 was adjusted to pH 9.5, and the reaction was carried out at 24℃ for 10 h for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 9.5 and pH 7.4 for 48 h in sequence.

[0044] Preparation Example 3:

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

[0046] 1.6 parts by weight of polylysine and 1.2 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 32°C for 1.6 h to obtain a mixture. The mixture obtained above and 4 parts by weight of L-cysteine ​​were stirred at 24°C for 36 h under nitrogen protection to carry out the first stirring reaction. After the reaction was completed, modified polylysine was obtained by dialysis and freeze-drying. 2.5 parts by weight of modified polylysine and 3 parts by weight of hexahydrophthalic anhydride were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of 4:1 was adjusted to pH 9.8, and the reaction was carried out at 24℃ for 14 hours for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 9.5 and pH 7.4 for 48 hours in sequence.

[0047] Preparation Example 4:

[0048] The preparation of polylysine complex includes the following steps:

[0049] Two parts by weight of polylysine and 1.5 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 35°C for 2 hours to obtain a mixture. The mixture obtained above and 5 parts by weight of L-cysteine ​​were stirred at 25°C for 48 hours under nitrogen protection to carry out the first stirring reaction. After the reaction was completed, modified polylysine was obtained by dialysis and freeze-drying. 3.5 parts by weight of modified polylysine and 4 parts by weight of hexahydrophthalic anhydride were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of 4:1 was adjusted to pH 10, and the reaction was carried out at 25°C for 16 hours for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 10 and pH 7.4 for 48 hours in sequence.

[0050] Preparation Example 5:

[0051] The preparation of polylysine complex includes 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 The ratio of 4:1 was adjusted to pH 10, and the reaction was carried out at 25°C for 16 hours for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 10 and pH 7.4 for 48 hours in sequence.

[0053] Preparation Example 6:

[0054] The preparation of polylysine complex includes the following steps:

[0055] Two parts by weight of polylysine and 1.5 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 35°C for 2 hours to obtain a mixture. The mixture obtained above and 5 parts by weight of L-cysteine ​​were stirred at 25°C for 48 hours under nitrogen protection to carry out the first stirring reaction. After the reaction was completed, modified polylysine was obtained by dialysis and freeze-drying. 3.5 parts by weight of modified polylysine and 4 parts by weight of phthalic acid were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of 4:1 was adjusted to pH 10, and the reaction was carried out at 25°C for 16 hours for a second stirring reaction. After the reaction was completed, the polylysine complex was obtained by dialyzing in deionized water at pH 10 and pH 7.4 for 48 hours in sequence.

[0056] Preparation Example 7:

[0057] The preparation of the polyethyleneimine composite includes the following steps:

[0058] Two parts by weight of polyethyleneimine and 1.5 parts by weight of N,N-carbonyldiimidazole were dispersed in 100 parts by weight of dimethyl sulfoxide and activated at 35°C for 2 hours to obtain a mixture. The mixture obtained above and 5 parts by weight of L-cysteine ​​were stirred at 25°C for 48 hours under nitrogen protection to carry out the first stirring reaction. After the reaction was completed, the mixture was dialyzed and freeze-dried to obtain modified polyethyleneimine. 3.5 parts by weight of modified polyethyleneimine and 4 parts by weight of phthalic acid were dispersed in 300 parts by weight of mixed solvent (V 去离子水 V 乙醇 The ratio of polyethyleneimine to polyimide was 4:1. After adjusting the pH to 10, the reaction was carried out at 25°C for 16 hours for a second stirring reaction. After the reaction was completed, the polyimide was dialyzed in deionized water at pH 10 and pH 7.4 for 48 hours in sequence to obtain the polyethyleneimine complex.

[0059] Preparation Example 8:

[0060] The preparation of collagen peptide complex includes the following steps:

[0061] Two parts by weight of recombinant human type III collagen were dispersed in 40 parts by weight of deionized water. After incubation at 50°C for 10 min, 0.04 parts by weight of alkaline protease were added and the pH was adjusted to 8.5. The reaction was carried out at 45°C for 2 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 95°C for 10 min. After cooling to 23°C, the supernatant was collected by centrifugation at 4000 r / min for 20 min to obtain the enzymatic hydrolysis product. 0.04 parts by weight of papain was added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 6.0. The reaction was carried out at 45°C for 2 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 95℃ for 10 min, followed by cooling to 23℃ and centrifugation at 4000 r / min for 20 min. The supernatant was collected to obtain recombinant human collagen peptides. 3 parts by weight of (3-propanoyl)triphenylphosphine bromide and 0.8 parts by weight of allyloxy polyoxyethylene ether were dispersed in 150 parts by weight of petroleum ether, and 0.3 parts by weight of sodium bisulfate were added. The mixture was reacted at 70℃ for 4 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. 1 part by weight of the unsaturated cationic compound and 0.5 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 V N,N-二甲基甲酰胺 In a 4:1 mixture of solvents, a second mixing reaction was carried out at 30°C for 48 h. After the reaction, the modified collagen peptides were purified by extraction, ion exchange chromatography, and freeze-drying. Two parts by weight of the modified recombinant human collagen peptides and four parts by weight of the polylysine complex prepared in Example 1 were dispersed in 200 parts by weight of deionized water and reacted at 23°C for 24 h to carry out a third mixing reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze-drying.

[0062] Preparation Example 9:

[0063] The preparation of collagen peptide complex includes the following steps:

[0064] 2.3 parts by weight of recombinant human type III collagen were dispersed in 45 parts by weight of deionized water. After incubation at 55°C for 12 min, 0.045 parts by weight of alkaline protease were added and the pH was adjusted to 9. The reaction was carried out at 46°C for 2.2 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 97°C for 12 min. After cooling to 24°C, the supernatant was collected by centrifugation at 4000 r / min for 24 min to obtain the enzymatic hydrolysis product. 0.043 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 6.3. The reaction was carried out at 47°C for 2.1 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 98℃ for 12 min, followed by cooling to 24℃ and centrifugation at 4000 r / min for 23 min. The supernatant was collected to obtain recombinant human collagen peptides. 3.2 parts by weight of (3-propanoyl)triphenylphosphine bromide and 0.9 parts by weight of allyloxy polyoxyethylene ether were dispersed in 180 parts by weight of petroleum ether, and then 0.32 parts by weight of sodium bisulfate were added. The mixture was reacted at 73℃ for 4.3 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. 1 part by weight of the unsaturated cationic compound and 0.7 parts 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 In a 4:1) mixed solvent, a second mixed reaction was carried out at 32°C for 54 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 2.5 parts by weight of the modified recombinant human collagen peptide and 4.3 parts by weight of the polylysine complex prepared in Example 2 were dispersed in 200 parts by weight of deionized water and reacted at 24°C for 26 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0065] Preparation Example 10:

[0066] The preparation of collagen peptide complex includes the following steps:

[0067] 2.6 parts by weight of recombinant human type III collagen were dispersed in 50 parts by weight of deionized water. After incubation at 56°C for 13 min, 0.052 parts by weight of alkaline protease were added and the pH was adjusted to 9.1. The reaction was carried out at 48°C for 2.6 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 98°C for 12 min. After cooling to 24°C, the supernatant was collected by centrifugation at 4000 r / min for 26 min to obtain the enzymatic hydrolysis product. 0.05 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 6.7. The reaction was carried out at 48°C for 2.8 h for the second enzymatic hydrolysis reaction. The enzyme was then inactivated by treatment at 98℃ for 12 min, cooled to 24℃, and centrifuged at 4000 r / min for 28 min to collect the supernatant, yielding recombinant human collagen peptides. 3.5 parts by weight of (3-propanoyl)triphenylphosphine bromide and 1 part by weight of allyloxy polyoxyethylene ether were dispersed in 250 parts by weight of petroleum ether, and then 0.38 parts by weight of sodium bisulfate were added. The mixture was reacted at 78℃ for 4.8 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. 1 part by weight of the unsaturated cationic compound and 0.7 parts 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 In a 4:1 mixture of solvents, a second mixing reaction was carried out at 34°C for 64 h. After the reaction, the modified collagen peptides were purified by extraction, ion exchange chromatography, freeze drying, and other steps. 2.8 parts by weight of the modified recombinant human collagen peptides and 4.6 parts by weight of the polylysine complex prepared in Example 3 were dispersed in 200 parts by weight of deionized water and reacted at 24°C for 28 h to carry out a third mixing reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0068] Preparation Example 11:

[0069] The preparation of collagen peptide complex includes the following steps:

[0070] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 100℃ for 15 min, followed by cooling to 25℃ and centrifugation at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine 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 were added. The mixture was reacted at 80℃ for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 V N,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Example 4 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0071] Preparation Example 12:

[0072] The preparation of collagen peptide complex includes the following steps:

[0073] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 100℃ for 15 min, followed by cooling to 25℃ and centrifugation at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine 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 were added. The mixture was reacted at 80℃ for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 V N,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Example 5 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0074] Preparation Example 13:

[0075] The preparation of collagen peptide complex includes the following steps:

[0076] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 100℃ for 15 min, followed by cooling to 25℃ and centrifugation at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine 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 were added. The mixture was reacted at 80℃ for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 V N,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Example 6 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0077] Preparation Example 14:

[0078] The preparation of collagen peptide complex includes the following steps:

[0079] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 100℃ for 15 min, followed by cooling to 25℃ and centrifugation at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine 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 were added. The mixture was reacted at 80℃ for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 V N,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptides were purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptides and 5 parts by weight of the polyethyleneimine complex prepared in Example 7 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0080] Preparation Example 15:

[0081] The preparation of collagen peptide complex includes the following steps:

[0082] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether were dispersed in 300 parts by weight of petroleum ether. Then, 0.4 parts by weight of sodium bisulfate were added and the reaction was carried out at 80°C for 5 h for the first mixing reaction. After the reaction, the unsaturated cationic compound was obtained by rotary evaporation, washing, and recrystallization. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V 去离子水 VN,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Example 4 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0083] Preparation Example 16:

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

[0085] Three parts by weight of recombinant human type III collagen were dispersed in 60 parts by weight of deionized water. After incubation at 60°C for 15 min, 0.06 parts by weight of papain were added and the pH was adjusted to 7.0. The mixture was then reacted at 50°C for 3 h for enzymatic hydrolysis. After the reaction, the mixture was treated at 100°C for 15 min to inactivate the enzyme. After cooling to 25°C, the mixture was centrifuged at 4000 r / min for 30 min, and the supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine bromide and 1.2 parts by weight of allyloxy polyoxyethylene ether were dispersed in 300 parts by weight of petroleum ether. Then, 0.4 parts by weight of sodium bisulfate were added and the mixture was reacted at 80°C for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V 去离子水 V N,N-二甲基甲酰胺 In a 4:1) mixed solvent, a second mixed reaction was carried out at 35°C for 72 h. After the reaction, the modified collagen peptide was purified by extraction, ion exchange chromatography, freeze drying and other steps. 3 parts by weight of the modified recombinant human collagen peptide and 5 parts by weight of the polylysine complex prepared in Example 4 were dispersed in 200 parts by weight of deionized water and reacted at 25°C for 30 h to carry out a third mixed reaction. After the reaction, the collagen peptide complex was obtained by dialysis and freeze drying.

[0086] Preparation Example 17:

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

[0088] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The mixture was reacted at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the mixture was treated at 100°C for 15 min to inactivate the enzyme. After cooling to 25°C, the mixture was centrifuged at 4000 r / min for 30 min, and the supernatant was collected to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The mixture was reacted at 50°C for 3 h for the second enzymatic hydrolysis reaction. After the reaction, the mixture was treated at 100°C for 15 min to inactivate the enzyme. After cooling to 25°C, the mixture was centrifuged at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides; 4 parts by weight of (3-propanoyl)triphenylphosphine bromide, 2.5 parts by weight of 1,3-dicyclohexylcarbodiimide and 2 parts by weight of N-hydroxysuccinimide were dispersed in 100 parts by weight of N,N-dimethylformamide and stirred at 60°C for 4 h, then 0.8 parts by weight of recombinant human collagen peptides were added and stirred at 23°C for 24 h. After the reaction was completed, the modified collagen peptides were obtained by rotary evaporation and dialysis; 3 parts by weight of modified recombinant human collagen peptides and 5 parts by weight of the polylysine complex obtained in Preparation Example 4 were 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 was completed, the collagen peptide complex was obtained by dialysis and freeze-drying.

[0089] Preparation Example 18:

[0090] The preparation of modified collagen peptides includes the following steps:

[0091] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. Enzyme inactivation was achieved by treatment at 100℃ for 15 min, followed by cooling to 25℃ and centrifugation at 4000 r / min for 30 min. The supernatant was collected to obtain recombinant human collagen peptides. Four parts by weight of (3-propanoyl)triphenylphosphine 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 were added. The mixture was reacted at 80℃ for 5 h for the first mixed reaction. After the reaction, the mixture was washed by rotary evaporation and recrystallized to obtain an unsaturated cationic compound. One part by weight of the unsaturated cationic compound and 0.8 parts by weight of recombinant human collagen peptides were dispersed in 50 parts by weight of (V... 去离子水 VN,N-二甲基甲酰胺 The modified collagen peptides were prepared by reacting the peptides in a 4:1 mixture of solvents at 35°C for 72 hours. After the reaction was completed, the peptides were purified by extraction, ion exchange chromatography, freeze drying and other steps.

[0092] Preparation Example 19:

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

[0094] Three parts by weight of recombinant human type III collagen were 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 were added and the pH was adjusted to 9.5. The reaction was carried out at 50°C for 3 h for the first enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the enzymatic hydrolysis product. 0.06 parts by weight of papain were added to 100 parts by weight of the enzymatic hydrolysis product and the pH was adjusted to 7.0. The reaction was carried out at 50°C for 3 h for the second enzymatic hydrolysis reaction. After the reaction, the enzyme was inactivated by treatment at 100°C for 15 min. After cooling to 25°C, the supernatant was collected by centrifugation at 4000 r / min for 30 min to obtain the recombinant human collagen peptide.

[0095] Example 1:

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

[0097] Two parts by weight of the collagen peptide complex prepared in Example 8, four parts by weight of carbomer 491 and 1.5 parts by weight of Cuban balsam oil were dispersed in 100 parts by weight of purified water and stirred at 35°C for 30 min to obtain a mixed emulsion. Then, one part by weight of sodium alginate was added to the mixed emulsion, and the mixture was heated to 50°C and stirred for 30 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0098] Example 2:

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

[0100] 2.3 parts by weight of the collagen peptide complex prepared in Example 9, 5 parts by weight of carbomer 981 and 1.8 parts by weight of Cuban balsam oil were dispersed in 110 parts by weight of purified water and stirred at 38°C for 35 min to obtain a mixed emulsion. Then, 1.4 parts by weight of sodium alginate were 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 collagen peptides.

[0101] Example 3:

[0102] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0103] 2.6 parts by weight of the collagen peptide complex prepared in Example 10, 6.5 parts by weight of carbomer 2020 and 2.1 parts by weight of Cuban balsam oil were dispersed in 115 parts by weight of purified water and stirred at 42°C for 45 min to obtain a mixed emulsion. Then, 1.6 parts by weight of sodium alginate were added to the mixed emulsion, and the mixture was heated to 55°C and stirred for 25 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0104] Example 4:

[0105] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0106] Three parts by weight of the collagen peptide complex prepared in Example 11, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0107] Comparative Example 1:

[0108] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0109] Three parts by weight of the collagen peptide complex prepared in Example 12, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0110] Comparative Example 2:

[0111] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0112] Three parts by weight of the collagen peptide complex prepared in Example 13, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0113] Comparative Example 3:

[0114] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0115] Three parts by weight of the collagen peptide complex prepared in Example 14, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0116] Comparative Example 4:

[0117] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0118] Three parts by weight of the collagen peptide complex prepared in Example 15, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0119] Comparative Example 5:

[0120] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0121] Three parts by weight of the collagen peptide complex prepared in Example 16, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0122] Comparative Example 6:

[0123] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0124] Three parts by weight of the collagen peptide complex prepared in Example 17, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0125] Comparative Example 7:

[0126] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes 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 Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Then, 2 parts by weight of sodium alginate were added to the mixed emulsion, and the temperature was raised to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0128] Comparative Example 8:

[0129] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0130] Three parts by weight of the collagen peptide complex prepared in Example 12 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Then, 2 parts by weight of sodium alginate were added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0131] Comparative Example 9:

[0132] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0133] Three parts by weight of the collagen peptide complex prepared in Example 12 and eight parts by weight of carbomer 981 were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0134] Comparative Example 10:

[0135] The preparation of a sustained-release gynecological gel containing recombinant human collagen peptides includes the following steps:

[0136] Three parts by weight of collagen peptides prepared in Example 19, eight parts by weight of carbomer 981 and 2.5 parts by weight of Cuban balsam oil were dispersed in 120 parts by weight of purified water and stirred at 45°C for 50 min to obtain a mixed emulsion. Two parts by weight of sodium alginate were then added to the mixed emulsion, and the mixture was heated to 60°C and stirred for 20 min to obtain a sustained-release gynecological gel containing recombinant human collagen peptides.

[0137] Performance testing:

[0138] The performance of the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Examples 1-4 and Comparative Examples 1-10 of the present invention was tested, and the test results are as follows.

[0139] Test Example 1: Stimulation 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 control group, respectively. Groups 1-14 were injected daily with 2 mL of the sustained-release gynecological gel containing recombinant human collagen peptides prepared in Examples 1-4 and Comparative Examples 1-10 for 7 consecutive days. The control group received no treatment. The rabbits' vaginas were observed daily for redness, swelling, or erosion. The severity of irritation was assessed using the following criteria: no redness - 0 points, slight redness - 1 point, severe redness - 2 points, mild erosion - 3 points, and severe erosion - 4 points. The daily scores were summarized and statistically analyzed, as shown in Table 1 below.

[0141] Table 1. Results of the irritation test

[0142]

[0143] The experimental results in Table 1 show that the sustained-release gynecological gel containing recombinant human collagen peptides prepared in this invention has low irritation and good safety.

[0144] Experimental Example 2: Animal Experiment

[0145] Healthy female rats were selected to establish a comprehensive vaginitis model. Three days before applying the experimental sample gel, estradiol benzoate was injected subcutaneously into the rats daily in the morning at a dose of 0.5 mg / rat / time. Streptomycin (50 μg / rat / time) was also injected intravaginally. Then, a mixed bacterial culture (1×10⁸ CFU / mL) consisting of Gardnerella vaginalis, Candida albicans, and Group B Streptococcus was injected intravaginally. All injections were administered once daily for three consecutive days. The control group rats were inoculated vaginally with 50 μL of strain-free PBS. Five days later, the rats' vaginas were observed. Successful modeling was indicated by redness, swelling, and thickening of the vulva and vagina, and a change in discharge to a cheesy or curd-like consistency. Forty-eight successfully modeled rats were randomly divided into 16 groups of three rats each. Groups 1-4 correspond to Examples 1-4, groups 5-14 correspond to Comparative Examples 1-10, group 15 corresponds to the positive control group, and group 16 is the negative control group. Group 16 consists of healthy rats that did not undergo the comprehensive vaginitis modeling treatment. Groups 1-14 were treated with the sustained-release gynecological gel containing recombinant human collagen peptides prepared in Examples 1-4 and Comparative Examples 1-10, respectively, applied once daily at a dosage of 0.5 g / kg body weight for 7 consecutive days. Group 15, the positive control group, was treated with the same amount of sterile saline solution once daily for 7 consecutive days. Seven days later, rats from Examples 1-4, Comparative Examples 1-10, the positive control group, and the negative control group were sacrificed. Vaginal tissue from the rats was then homogenized, centrifuged at 4000 r / min for 10 min, and the precipitate was discarded. The levels of IL-6 and IL-8 in the vaginal tissue of rats from Examples 1-4, Comparative Examples 1-10, the positive control group, and the negative control group were detected using an ELISA kit. The reduction rate (%) of IL-6 and IL-8 in Examples 1-4 and Comparative Examples 1-10 was calculated. The experimental results were averaged and are shown in Table 2.

[0146] Reduction rate (%) = (M1-M0) / M0×100%, where M1 is the IL-6 or IL-8 content of Examples 1-4, Comparative Examples 1-10 and the positive control group; M0 is the IL-6 or IL-8 content of the negative control group;

[0147] Table 2. Animal Experiment Results

[0148]

[0149] As can be observed from the experimental results in Table 2, the sustained-release gynecological gel containing recombinant human collagen peptides prepared in Examples 1-4 of this invention can effectively reduce the content of inflammatory factors IL-6 or IL-8 in rat vaginal tissue, have good antibacterial and anti-inflammatory effects, repair vaginal damage, and effectively promote the treatment effect of gynecological diseases such as vaginitis.

[0150] Experimental Example 3: In vitro release effect detection

[0151] 10 mg of the sustained-release gynecological gel containing recombinant human collagen peptides prepared in Examples 1-4 and Comparative Examples 1, 2, 3, 7, and 10 were weighed and added to 50 mL centrifuge tubes. The tubes were then immersed in 30 mL of PBS solutions with pH=3.5, pH=5.0, and pH=7.0, respectively, and placed in a constant temperature shaker at 100 rpm and 37 °C. At different times, 3 nL of supernatant was taken out and replaced with an equal volume of fresh PBS solution. The concentration of recombinant human collagen peptides was determined by UV-Vis spectrophotometry. The results are shown in Tables 3, 4, and 5.

[0152] Table 3. In vitro release effect test (pH=3.5)

[0153]

[0154] Table 4. In vitro release effect test (pH=5.0)

[0155]

[0156] Table 5. In vitro release effect test (pH=7.4)

[0157]

[0158] The experimental results in Tables 3, 4, and 5 show that the sustained-release gynecological gels containing recombinant human collagen peptides prepared in Examples 1-4 of this invention exhibit good responsiveness and sustained-release performance in the inflammatory microenvironment, with stable sustained-release effects. This is beneficial for repairing damaged skin barriers, extending the treatment cycle, and improving the repair effect of damaged cells. In the normal acidic environment of the vagina, the modified collagen peptides are rapidly released; in the slightly acidic environment of vaginal inflammation, a responsive sustained-release effect is observed. The sustained-release gynecological gels containing recombinant human collagen peptides prepared in Comparative Examples 1, 2, 3, 7, and 10 do not exhibit the same sustained-release effect as Examples 1-4, making it difficult to achieve long-term treatment.

[0159] The embodiments described above provide a detailed explanation of the technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A method for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide, characterized by, The preparation method comprises the following steps: The preparation method of the collagen peptide complex comprises the following steps: The recombinant human collagen is sequentially subjected to first enzymolysis reaction by alkaline protease and second enzymolysis reaction by papain to obtain recombinant human collagen peptide; (3-propylcarboxyl) triphenylphosphonium bromide, allyloxy polyoxyethylene ether, sodium bisulfate and petroleum ether are subjected to first mixing reaction to obtain an unsaturated bond cationic compound; the unsaturated bond cationic compound and the recombinant human collagen peptide are subjected to second mixing reaction to obtain modified recombinant human collagen peptide; and the modified recombinant human collagen peptide and a polylysine complex are subjected to third mixing reaction to obtain the collagen peptide complex. 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 first stirring reaction to obtain modified polylysine, and the modified polylysine and hexahydrophthalic anhydride are subjected to second stirring reaction to obtain the polylysine complex. The recombinant human collagen is recombinant human type III collagen. 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.

2. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The weight ratio of the unsaturated bond cationic compound and the recombinant human collagen peptide is 1:0.5-0.

8.

3. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The weight ratio of the modified recombinant human collagen peptide and the polylysine complex is 2-3:4-5.

4. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The weight ratio of the polylysine, N,N-carbonyldiimidazole and L-cysteine is 1-2:0.5-1.5:2-5.

5. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The weight ratio of the modified polylysine and hexahydrophthalic anhydride is 1.5-3.5:2-4.

6. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The weight ratio of the collagen peptide complex, carbomer, Cuban balsam oil, purified water and sodium alginate is 2-3:4-8:1.5-2.5:100-120:1-2.

7. A process for the preparation of a sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide as claimed in claim 1, wherein, The preparation method is prepared by any one of claims 1-7.

8. A sustained release gynecological gel for vaginitis containing recombinant human-derived collagen peptide, characterized by, ​

Images