Long-acting bacteriostatic complex lysozyme gynecological lotion and preparation method thereof

Abstract

This invention relates to a long-acting antibacterial compound lysozyme gynecological wash and its preparation method, belonging to the technical field of gynecological care products. The gynecological wash formula is as follows (by weight): 0.5-5.0 parts of a nanogel complex loaded with modified lysozyme, 0.1-0.8 parts of recombinant human collagen, 0.3-1.5 parts of inulin, 0.3-1.0 parts of chitosan, 0.2-0.8 parts of sodium alginate, 0.1-0.5 parts of hydroxypropyl methylcellulose, 2.0-8.0 parts of glycerin, 1.0-5.0 parts of propylene glycol, 0.1-0.5 parts of lactic acid, 0.05-0.2 parts of citric acid, 0.02-0.1 parts of EDTA-2Na, 0.3-0.8 parts of phenoxyethanol, and 80-90 parts of purified water. Gallic acid modification broadens the antibacterial spectrum of lysozyme and improves its stability and resistance to protease degradation during storage and use. Encapsulating the modified lysozyme in the nanogel achieves a sustained-release effect in the vaginal environment, prolonging the antibacterial duration. The gynecological wash prepared by this invention has the characteristic of long-lasting antibacterial effect.

Description

Technical Field

[0001] This invention belongs to the field of gynecological care product technology, and relates to a long-acting antibacterial compound lysozyme gynecological wash and its preparation method. Background Technology

[0002] Bacterial vaginosis, vulvovaginal candidiasis, and other gynecological inflammations are common and frequently occurring diseases in women, and their occurrence is closely related to the imbalance of the vaginal microecology. Lysozyme, as a natural antibacterial enzyme, can hydrolyze the mucopolysaccharides in bacterial cell walls. It has the advantages of not producing drug resistance and being mild and non-irritating, and has been widely used in gynecological wash products in recent years.

[0003] However, existing lysozyme gynecological washes have the following technical defects: lysozyme has poor stability in liquid environments, especially its ability to inhibit Gram-negative bacteria is limited; most washes are rinse-type, and the effective ingredients are difficult to remain on the vaginal mucosa surface and are quickly lost after use, resulting in a short duration of antibacterial activity; they lack the ability to repair damaged mucosa, and simply killing bacteria is not conducive to the comprehensive restoration of the vaginal microecology; they cannot regulate the beneficial bacteria in the vagina, which can easily cause secondary imbalance of the flora.

[0004] Therefore, there is an urgent need to develop a long-acting antibacterial compound lysozyme gynecological wash and its preparation method. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a long-acting antibacterial compound lysozyme gynecological wash and its preparation method.

[0006] The objective of this invention can be achieved through the following technical solutions: A long-acting antibacterial compound lysozyme gynecological wash, the formula of which is as follows (by weight): 0.5-5.0 parts of a nanogel complex loaded with modified lysozyme, 0.1-0.8 parts of recombinant human collagen, 0.3-1.5 parts of inulin, 0.3-1.0 parts of chitosan, 0.2-0.8 parts of sodium alginate, 0.1-0.5 parts of hydroxypropyl methylcellulose, 2.0-8.0 parts of glycerin, 1.0-5.0 parts of propylene glycol, 0.1-0.5 parts of lactic acid, 0.05-0.2 parts of citric acid, 0.02-0.1 parts of EDTA-2Na, 0.3-0.8 parts of phenoxyethanol, and 80-90 parts of purified water.

[0007] As a preferred embodiment of the present invention, the preparation method of the nanogel complex loaded with modified lysozyme is as follows: S2-1: Gallic acid is dissolved in a 0.1M NaOH solution, the pH is adjusted to 5.0-6.0, carbodiimide is added and stirred for 15-30 min, followed by egg white lysozyme. The mass ratio of carbodiimide to lysozyme is (1-2):1. The mixture is stirred in a water bath at 30-40℃ for 4-8 h, and after dialysis and freeze-drying, modified lysozyme is obtained. S2-2: Methacrylic acid and polyethylene glycol diacrylate were mixed and dissolved in ethanol. N,N'-bis(acryloyl)cysteine ​​and azobisisobutyronitrile were added, wherein the mass ratio of N,N'-bis(acryloyl)cysteine ​​to methacrylic acid was (0.05~0.2):1, and the mass ratio of azobisisobutyronitrile to methacrylic acid was (0.005~0.02):1. The mixture was stirred at 75~85℃ for 4~6h under nitrogen protection. After centrifugation, dialysis, and freeze-drying, a nanogel carrier was obtained. S2-3: Disperse the nanogel carrier in purified water, add modified lysozyme, stir and load at 4°C for 12-24 hours, and then centrifuge and freeze dry to obtain the nanogel complex loaded with modified lysozyme.

[0008] As a preferred embodiment of the present invention, the mass ratio of gallic acid to egg white lysozyme in S2-1 is 1:(2~5).

[0009] As a preferred embodiment of the present invention, in S2-2, methacrylic acid and polyethylene glycol diacrylate are mixed at a mass ratio of (2~4):1.

[0010] As a preferred embodiment of the present invention, the mass ratio of the nanogel carrier to the modified lysozyme in S2-3 is (1.5~2.5):1.

[0011] As a preferred embodiment of the present invention, the recombinant human collagen is recombinant human type III collagen with a molecular weight of 50-120 kDa.

[0012] As a preferred embodiment of the present invention, the inulin is a low-polymerization inulin with an average degree of polymerization ≤10.

[0013] As a preferred embodiment of the present invention, the chitosan has a molecular weight of 50,000 to 200,000 and a degree of deacetylation of ≥90%.

[0014] A method for preparing a long-acting antibacterial compound lysozyme gynecological wash, the specific steps of which are as follows: S9-1: Take half the mass of purified water according to the formula ratio, heat to 40~50℃, add sodium alginate while stirring, disperse evenly and let stand for 12~24h; S9-2: Add chitosan and hydroxypropyl methylcellulose sequentially, and stir for 30-60 min; S9-3: Add glycerol and propylene glycol, stir until homogeneous, cool to 30~40℃, add the nanogel complex loaded with modified lysozyme, and stir until uniformly dispersed; S9-4: Add recombinant human collagen and inulin in sequence, and stir to dissolve; S9-5: Add EDTA-2Na and phenoxyethanol; S9-6: Adjust the pH of the system to 4.0~4.5 using lactic acid and citric acid; S9-7: Add the remaining purified water, continue stirring for 15-30 minutes, then homogenize, fill and seal to obtain the finished product.

[0015] As a preferred embodiment of the present invention, the homogenization process is carried out at a rotation speed of 8000~10000 rpm for a time of 10~15 min.

[0016] The gallic acid-modified egg white lysozyme was prepared by covalently binding gallic acid and egg white lysozyme under carbodiimide catalysis. Gallic acid has antibacterial and antioxidant activities, and its covalent binding with lysozyme not only enhances the antibacterial ability of lysozyme against Gram-negative bacteria, but also improves the enzyme's thermal stability and resistance to protease degradation during storage and use.

[0017] The nanogel is a nanogel with a framework of methacrylic acid and polyethylene glycol diacrylate, containing a disulfide bond cross-linking structure, and internally loaded with gallic acid-modified egg white lysozyme. This nanogel exhibits dual-response characteristics: in the normal acidic environment of the vagina, the nanogel structure is compact, and lysozyme is released slowly; when the vaginal pH rises above 5.5, indicating inflammation or dysbiosis, the nanogel structure relaxes, accelerating the release of lysozyme. Simultaneously, increased levels of reactive oxygen species (ROS) at the site of inflammation can break disulfide bonds, further triggering the on-demand release of lysozyme.

[0018] The recombinant human collagen is preferably recombinant human type III collagen, which can promote the adhesion, proliferation and migration of vaginal epithelial cells, repair minor mucosal damage caused by inflammation or mechanical flushing, and restore the local physical barrier function.

[0019] The inulin is a low-polymerization inulin, which, as a prebiotic, can selectively promote the proliferation of endogenous lactobacilli in the vagina, maintain a normal acidic pH environment, and achieve microecological regulation.

[0020] The beneficial effects of this invention are: This invention significantly enhances the inhibitory effect against Gram-negative bacteria by covalently modifying egg white lysozyme with gallic acid, while also improving the thermostability and storage stability of lysozyme, overcoming the shortcomings of ordinary lysozyme such as narrow antibacterial spectrum and easy inactivation. The lysozyme is modified by loading it onto a nanogel, enabling on-demand drug release within the vaginal environment. It releases slowly under normal acidic conditions to maintain basic protection, and accelerates release when inflammation causes pH elevation or oxidative stress, significantly extending the effective antibacterial duration of a single application. The introduction of recombinant human collagen can actively repair minor damage to the vaginal mucosa, restore the integrity of the epithelial barrier, and reduce the chance of pathogen colonization, making it particularly suitable for patients with recurrent gynecological inflammation. Inulin, as a prebiotic, selectively promotes the proliferation of beneficial lactobacilli in the vagina, maintaining a normal acidic pH environment, achieving a virtuous cycle of inhibition-repair-promoting bacteria. Detailed Implementation

[0021] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0022] It should be noted that, unless otherwise specified, the present invention does not specifically limit the source of the raw materials used in the following embodiments. Commercially available products or products prepared by conventional preparation methods that are well known to those skilled in the art can be used. Experimental methods that do not specify specific conditions are all conventional methods and conventional conditions well known in the art.

[0023] Example 1

[0024] The formula of the gynecological wash is as follows, calculated by weight: 2.5 parts of a nanogel complex loaded with gallic acid-modified lysozyme, 0.4 parts of recombinant human type III collagen (molecular weight 80kDa, purity ≥95%), 0.8 parts of inulin (low degree of polymerization, average degree of polymerization 8), 0.6 parts of chitosan (molecular weight 100,000, degree of deacetylation 92%), 0.5 parts of sodium alginate, 0.3 parts of hydroxypropyl methylcellulose, 5.0 parts of glycerin, 3.0 parts of propylene glycol, 0.3 parts of lactic acid, 0.1 parts of citric acid, 0.05 parts of EDTA-2Na, 0.5 parts of phenoxyethanol, and 85 parts of purified water.

[0025] The preparation method of the nanogel complex loaded with modified lysozyme is as follows: S2-1: Gallic acid was dissolved in a 0.1M NaOH solution, the pH was adjusted to 5.5, carbodiimide was added and stirred for 25 min, followed by egg white lysozyme. The mass ratio of carbodiimide to lysozyme was 1.5:1, and the mass ratio of gallic acid to egg white lysozyme was 1:3.5. The mixture was stirred in a water bath at 35℃ for 6 h, and after dialysis and freeze-drying, modified lysozyme was obtained. S2-2: Methacrylic acid and polyethylene glycol diacrylate were mixed at a mass ratio of 3:1 and dissolved in ethanol. N,N'-bis(acryloyl)cysteine ​​and azobisisobutyronitrile were added, wherein the mass ratio of N,N'-bis(acryloyl)cysteine ​​to methacrylic acid was 0.1:1 and the mass ratio of azobisisobutyronitrile to methacrylic acid was 0.01:1. The mixture was stirred at 80°C for 5 hours under nitrogen protection, and then centrifuged, dialyzed, and freeze-dried to obtain a nanogel carrier. S2-3: Disperse the nanogel carrier in purified water, add modified lysozyme, the mass ratio of nanogel carrier to modified lysozyme is 2:1, stir and load at 4℃ for 18h, and then centrifuge and freeze dry to obtain the nanogel complex loaded with modified lysozyme.

[0026] Example 2

[0027] The only difference between this embodiment and Example 1 is that the preparation method of the nanogel complex loaded with modified lysozyme is as follows. S2-1: Gallic acid was dissolved in a 0.1M NaOH solution, the pH was adjusted to 5.0, carbodiimide was added and stirred for 15 min, followed by egg white lysozyme. The mass ratio of carbodiimide to lysozyme was 1:1, and the mass ratio of gallic acid to egg white lysozyme was 1:2. The mixture was stirred in a water bath at 30℃ for 4 h, and after dialysis and freeze-drying, modified lysozyme was obtained. S2-2: Methacrylic acid and polyethylene glycol diacrylate were mixed at a mass ratio of 2:1 and dissolved in ethanol. N,N'-bis(acryloyl)cysteine ​​and azobisisobutyronitrile were added, wherein the mass ratio of N,N'-bis(acryloyl)cysteine ​​to methacrylic acid was 0.05:1 and the mass ratio of azobisisobutyronitrile to methacrylic acid was 0.005:1. The mixture was stirred at 75°C for 4 hours under nitrogen protection, and then centrifuged, dialyzed, and freeze-dried to obtain a nanogel carrier. S2-3: Disperse the nanogel carrier in purified water, add modified lysozyme, the mass ratio of nanogel carrier to modified lysozyme is 1.5:1, stir and load at 4℃ for 12h, then centrifuge and freeze dry to obtain the nanogel complex loaded with modified lysozyme.

[0028] Everything else is the same as in Example 1.

[0029] Example 3

[0030] The only difference between this embodiment and Example 1 is that the preparation method of the nanogel complex loaded with modified lysozyme is as follows. S2-1: Gallic acid was dissolved in a 0.1M NaOH solution, the pH was adjusted to 6.0, carbodiimide was added and stirred for 30 min, followed by egg white lysozyme. The mass ratio of carbodiimide to lysozyme was 2:1, and the mass ratio of gallic acid to egg white lysozyme was 1:5. The mixture was stirred in a water bath at 40℃ for 8 h, and after dialysis and freeze-drying, modified lysozyme was obtained. S2-2: Methacrylic acid and polyethylene glycol diacrylate were mixed at a mass ratio of 4:1 and dissolved in ethanol. N,N'-bis(acryloyl)cysteine ​​and azobisisobutyronitrile were added, wherein the mass ratio of N,N'-bis(acryloyl)cysteine ​​to methacrylic acid was 0.2:1 and the mass ratio of azobisisobutyronitrile to methacrylic acid was 0.02:1. The mixture was stirred at 85°C for 6 hours under nitrogen protection, and then centrifuged, dialyzed, and freeze-dried to obtain a nanogel carrier. S2-3: Disperse the nanogel carrier in purified water, add modified lysozyme, the mass ratio of nanogel carrier to modified lysozyme is 2.5:1, stir and load at 4℃ for 24h, then centrifuge and freeze dry to obtain the nanogel complex loaded with modified lysozyme.

[0031] Everything else is the same as in Example 1.

[0032] Example 4

[0033] The difference between this embodiment and Example 1 is only that the gynecological wash formula is as follows, calculated by weight: 0.5 parts of a nanogel complex loaded with gallic acid-modified lysozyme, 0.1 parts of recombinant human type III collagen (molecular weight 80kDa, purity ≥95%), 0.3 parts of inulin (low degree of polymerization, average degree of polymerization 8), 0.3 parts of chitosan (molecular weight 100,000, degree of deacetylation 92%), 0.2 parts of sodium alginate, 0.1 parts of hydroxypropyl methylcellulose, 2.0 parts of glycerol, 1.0 part of propylene glycol, 0.1 parts of lactic acid, 0.2 parts of citric acid, 0.02 parts of EDTA-2Na, 0.3 parts of phenoxyethanol, and 80 parts of purified water.

[0034] Everything else is the same as in Example 1.

[0035] Example 5

[0036] The difference between this embodiment and Example 1 is only that the gynecological wash formula is as follows, calculated by weight: 5.0 parts of a nanogel complex loaded with gallic acid-modified lysozyme, 0.8 parts of recombinant human type III collagen (molecular weight 80kDa, purity ≥95%), 1.5 parts of inulin (low degree of polymerization, average degree of polymerization 8), 1.0 part of chitosan (molecular weight 100,000, degree of deacetylation 92%), 0.8 parts of sodium alginate, 0.5 parts of hydroxypropyl methylcellulose, 85.0 parts of glycerol, 5.0 parts of propylene glycol, 0.5 parts of lactic acid, 0.05 parts of citric acid, 0.1 parts of EDTA-2Na, 0.8 parts of phenoxyethanol, and 90 parts of purified water.

[0037] Everything else is the same as in Example 1.

[0038] Example 6

[0039] The only difference between this embodiment and Embodiment 1 is that the homogenization process in the preparation method of the long-acting antibacterial compound lysozyme gynecological wash is performed at a speed of 8000 rpm for 15 minutes.

[0040] Everything else is the same as in Example 1.

[0041] Example 7

[0042] The only difference between this embodiment and Embodiment 1 is that the homogenization process in the preparation method of the long-acting antibacterial compound lysozyme gynecological wash is performed at a speed of 10,000 rpm for 10 minutes.

[0043] Everything else is the same as in Example 1.

[0044] Comparative Example 1 The only difference between this comparative example and Example 1 is that untreated egg white lysozyme is used instead of the nanogel complex loaded with modified lysozyme; otherwise, they are the same as in Example 1.

[0045] Comparative Example 2 The only difference between this comparative example and Example 1 is that gallic acid was not added in the preparation of the nanogel complex loaded with modified lysozyme; otherwise, they are the same as in Example 1.

[0046] Comparative Example 3 The only difference between this comparative example and Example 1 is that the modified lysozyme is not subjected to nanogel loading treatment; otherwise, they are the same as in Example 1.

[0047] Antibacterial performance test The test strains used were *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, and *Candida albicans* ATCC 10231. The test samples were mixed with bacterial suspensions (1×10⁻⁶). 6Mix the CFU / mL solution thoroughly and incubate at 37 ℃ for 2 h, 12 h, and 48 h to determine the number of surviving bacteria. Compare the results with the blank control (physiological saline) to calculate the inhibition rate. The specific experimental results are recorded in the table below.

[0048] Table 1. Escherichia coli inhibition rate

[0049] Table 2. Staphylococcus aureus inhibition rate

[0050] Table 3. Antibacterial rate of Candida albicans

[0051] As can be seen from the above examples and comparative data, gallic acid modification broadens the antibacterial spectrum of lysozyme, and the nanogel further provides a long-lasting sustained-release antibacterial effect.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention are still within the scope of the present invention.

Claims

1. A long-lasting antibacterial compound lysozyme gynecological wash, characterized in that, The gynecological wash formula is as follows, calculated by weight: 0.5-5.0 parts of nanogel complex loaded with modified lysozyme, 0.1-0.8 parts of recombinant human collagen, 0.3-1.5 parts of inulin, 0.3-1.0 parts of chitosan, 0.2-0.8 parts of sodium alginate, 0.1-0.5 parts of hydroxypropyl methylcellulose, 2.0-8.0 parts of glycerin, 1.0-5.0 parts of propylene glycol, 0.1-0.5 parts of lactic acid, 0.05-0.2 parts of citric acid, 0.02-0.1 parts of EDTA-2Na, 0.3-0.8 parts of phenoxyethanol, and 80-90 parts of purified water.

2. The long-acting antibacterial compound lysozyme gynecological wash according to claim 1, characterized in that, The preparation method of the nanogel complex loaded with modified lysozyme is as follows: S2-1: Gallic acid is dissolved in a 0.1M NaOH solution, the pH is adjusted to 5.0-6.0, carbodiimide is added and stirred for 15-30 min, followed by egg white lysozyme. The mass ratio of carbodiimide to lysozyme is (1-2):

1. The mixture is stirred in a water bath at 30-40℃ for 4-8 h, and after dialysis and freeze-drying, modified lysozyme is obtained. S2-2: Methacrylic acid and polyethylene glycol diacrylate were mixed and dissolved in ethanol. N,N'-bis(acryloyl)cysteine ​​and azobisisobutyronitrile were added, wherein the mass ratio of N,N'-bis(acryloyl)cysteine ​​to methacrylic acid was (0.05~0.2):1, and the mass ratio of azobisisobutyronitrile to methacrylic acid was (0.005~0.02):

1. The mixture was stirred at 75~85℃ for 4~6h under nitrogen protection. After centrifugation, dialysis, and freeze-drying, a nanogel carrier was obtained. S2-3: Disperse the nanogel carrier in purified water, add modified lysozyme, stir and load at 4°C for 12-24 hours, and then centrifuge and freeze dry to obtain the nanogel complex loaded with modified lysozyme.

3. The long-acting antibacterial compound lysozyme gynecological wash according to claim 2, characterized in that, The mass ratio of gallic acid to egg white lysozyme in S2-1 is 1:(2~5).

4. The long-acting antibacterial compound lysozyme gynecological wash according to claim 2, characterized in that, In S2-2, methacrylic acid and polyethylene glycol diacrylate are mixed at a mass ratio of (2~4):

1.

5. The long-acting antibacterial compound lysozyme gynecological wash according to claim 2, characterized in that, The mass ratio of the nanogel carrier to the modified lysozyme in S2-3 is (1.5~2.5):

1.

6. The long-acting antibacterial compound lysozyme gynecological wash according to claim 1, characterized in that, The recombinant human collagen is recombinant human type III collagen with a molecular weight of 50-120 kDa.

7. The long-acting antibacterial compound lysozyme gynecological wash according to claim 1, characterized in that, The inulin is a low-polymerization inulin with an average degree of polymerization of ≤10.

8. The long-acting antibacterial compound lysozyme gynecological wash according to claim 1, characterized in that, The chitosan has a molecular weight of 50,000 to 200,000 and a degree of deacetylation of ≥90%.

9. A method for preparing a long-acting antibacterial compound lysozyme gynecological wash as described in any one of claims 1 to 8, characterized in that, The specific steps of the preparation method are as follows: S9-1: Take half the mass of purified water according to the formula ratio, heat to 40~50℃, add sodium alginate while stirring, disperse evenly and let stand for 12~24h; S9-2: Add chitosan and hydroxypropyl methylcellulose sequentially, and stir for 30-60 min; S9-3: Add glycerol and propylene glycol, stir until homogeneous, cool to 30~40℃, add the nanogel complex loaded with modified lysozyme, and stir until uniformly dispersed; S9-4: Add recombinant human collagen and inulin in sequence, and stir to dissolve; S9-5: Add EDTA-2Na and phenoxyethanol; S9-6: Adjust the pH of the system to 4.0~4.5 using lactic acid and citric acid; S9-7: Add the remaining purified water, continue stirring for 15-30 minutes, then homogenize, fill and seal to obtain the finished product.

10. The method for preparing a long-acting antibacterial compound lysozyme gynecological wash according to claim 9, characterized in that, The homogenization process is carried out at a speed of 8000~10000 rpm for 10~15 min.

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