Gynecological antibacterial gel containing carbomer component

Abstract

The invention discloses a gynecological antibacterial gel containing a carbomer component, and belongs to the technical field of gynecological nursing materials. The gel is prepared from 0.5 to 5 percent of plant extract microcapsules, 0.5 to 3 percent of carbomer, 2 to 10 percent of glycerol, 0.1 to 1.5 percent of triethanol and the balance of purified water. The plant extract microcapsule is prepared by compounding methacrylated silk fibroin and a caffeic acid hyaluronic acid graft, has temperature responsiveness, can improve the adhesive force on the surface of vaginal mucosa, and realizes slow release of the plant extract; the caffeic acid catechol hydroxyl is chelated with silver ions, a nano-silver monodisperse layer is formed on the surface of the microcapsule, and the antibacterial activity is improved. The composite plant extract can inhibit bacteria, repair mucous membranes and maintain the acidic microenvironment of the vagina. When the gel is prepared, the problem that the swelling efficiency of carbomer is low is solved through glycerol pre-grinding, and triethanolamine is added in three times to form a gradient cross-linked network. The problems that traditional gel is unstable in viscosity, poor in mucous membrane adhesiveness and prone to damaging vagina micro-ecology are solved, the antibacterial effect is good, and preservation is stable.

Description

Technical Field

[0001] The present invention belongs to the technical field of gynecological care products, and particularly relates to a bacteriostatic gel for gynecology containing carbomer components. Background Art

[0002] Gynecological inflammations, such as vaginitis, cervicitis, etc., are extremely common diseases among the female population. In the clinical treatment of such diseases, topical administration preparations play a key role, and dosage forms such as suppositories, creams, and gels are widely used. The gel dosage form, due to its excellent mucoadhesiveness, can adhere well to the vaginal mucosa surface, thereby prolonging the contact time between the drug and the lesion; it has a sustained-release property, enabling the drug to be released continuously and stably, maintaining an effective drug concentration; and because it is relatively convenient and comfortable to use, it is more easily accepted by patients, and thus has gradually become a research hotspot in this field.

[0003] However, the current gel dosage forms still have many problems to be solved urgently. In terms of viscosity stability, the vaginal environment is relatively special, with its temperature usually maintained at about 37°C, and the pH value fluctuating within the range of 3.8 - 5.0. Such environmental conditions are extremely likely to cause significant fluctuations in the viscosity of the gel. When the gel viscosity is unstable, it will seriously affect the residence time of the drug in the vagina. If the viscosity decreases, the gel is likely to flow out of the vagina and cannot act on the lesion site for a long time; if the viscosity increases, it may affect the release rate and uniformity of the drug. At the same time, the adhesiveness of the gel in the vagina is poor, which makes it difficult for the gel to closely adhere to the vaginal mucosa, and the drug is likely to be lost, greatly reducing the therapeutic effect of the drug. Some gels may damage the vaginal microecological balance due to too high a pH value or the selected excipients being irritating. There is a complex and delicate microbial community in the vagina, maintaining a specific ecological balance, which plays an important protective role in the health of the female reproductive system. Once the gel destroys this microecological balance, a series of adverse consequences may occur, such as a decrease in the number of beneficial bacteria in the vagina and a large reproduction of harmful bacteria, thereby increasing the risk of women being infected with other gynecological diseases, and even possibly leading to the aggravation of the original inflammation. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention prepared plant extract microcapsules and compounded them with a carbomer matrix, solving the problems of unstable viscosity, poor mucosal adhesiveness, and easy destruction of the vaginal microecology of traditional gynecological bacteriostatic gels.

[0005] In order to achieve the above object, the following technical solution is adopted: The present invention provides a bacteriostatic gel for gynecology containing carbomer components, which includes the following components in mass percentage: Plant extract microcapsules 0.5 - 5%, carbomer 0.5 - 3%, glycerol 2 - 10%, triethanolamine 0.1 - 1.5%, and the rest is purified water.

[0006] The preparation process of the plant extract microcapsules is as follows: S1. Boil silk in a 0.5% sodium carbonate solution by mass fraction for 1 h to obtain degummed silk. After drying, dissolve it in a 10 mol / L lithium bromide solution, then add glycidyl methacrylate, and react at 60 °C for 4 h under nitrogen protection. Dialyze the reaction solution with a filter membrane with a molecular weight cut-off of 8 kDa, and freeze-dry to obtain methacrylated silk fibroin. S2. Dissolve hyaluronic acid in a MES buffer solution with pH = 5.0, add EDC and NHS, and stir in the dark at room temperature for 2 h to form an active hyaluronic acid solution; dissolve caffeic acid in dimethyl sulfoxide, add HOBt and DIC, and stir at room temperature for 1 h to obtain an activated caffeic acid solution. Mix the active hyaluronic acid solution with the activated caffeic acid solution, adjust the pH to 7.4, and react in the dark at 50 °C for 24 h. Filter the reaction solution through a 0.45 μm filter membrane, dialyze it with a filter membrane with a molecular weight cut-off of 3.5 kDa, and freeze-dry to obtain a caffeic acid-hyaluronic acid graft. S3. Mix methacrylated silk fibroin and caffeic acid-hyaluronic acid graft, add them to an ethanol solution, add tartaric acid to adjust the pH to 5.0 - 5.5 to form a homogeneous solution. After mixing the composite plant extract and Tween 80, slowly add them to the homogeneous solution. Drop a 0.01% silver nitrate aqueous solution by mass concentration into the homogeneous solution, then add sodium sulfite, react in the dark at 50 °C for 30 min, and then add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate accounting for 0.25% of the mass of the homogeneous solution. Cure it with ultraviolet light, then centrifuge at 4000 rpm for 10 min to collect the microcapsules, wash them 3 times with deionized water, and freeze-dry to obtain plant extract microcapsules loaded with silver nanoparticles.

[0007] Further, the mass ratio of the degummed silk to the lithium bromide solution is 1:10 - 15, and the dosage of glycidyl methacrylate is 10 - 20% of the mass of silk fibroin.

[0008] Further, the mass of the MES buffer solution is 20 - 30 times that of hyaluronic acid; the molar amount of EDC is 1.5 - 1.8 times that of the carboxyl group of hyaluronic acid, and the molar amount of NHS is 0.5 - 0.8 times that of EDC; the mass of dimethyl sulfoxide is 20 - 30 times that of caffeic acid, and the molar amounts of HOBt, DIC, and caffeic acid are 1.0 - 1.2:1.5 - 1.8:1.

[0009] Further, the mass ratio of the active hyaluronic acid solution to the activated caffeic acid solution is 1:1.2 - 1.5.

[0010] Further, the mass ratio of methacrylated silk fibroin to caffeic acid-hyaluronic acid graft is 1:2.

[0011] Further, in step S3, the mass of the ethanol solution is 10 - 15 times that of the methacrylated silk fibroin and caffeic acid hyaluronic acid graft, and the mass fraction of the ethanol solution is 60 - 75%.

[0012] Further, the mass ratio of the composite plant extract to Tween 80 is 10 - 15:1; the mass of the silver nitrate aqueous solution is 5 - 10% of the homogenized solution, and the mass of sodium sulfite is 0.02 - 0.05% of the homogenized solution.

[0013] Further, the composite plant extract comprises the following components in parts by mass: 0.5 - 2 parts of bearberry leaf extract, acanthopanax senticosus extract, 1 - 3 parts of motherwort extract, 1 - 3 parts of witch hazel extract, 25 - 30 parts of zedoary essential oil, 3 - 5 parts of jojoba oil, and 2 - 4 parts of citronella oil.

[0014] The bearberry leaf extract contains arbutin, ursolic acid and flavonoid components, and has the effects of bacteriostasis and mucosal repair. Arbutin inhibits pathogenic tyrosinase and cell wall synthesis, and targets and inhibits pathogenic bacteria such as candida albicans; ursolic acid reduces mucosal redness and congestion and accelerates epithelial cell repair by regulating the inflammatory signaling pathway. Its flavonoid components can selectively promote the proliferation of lactobacilli and maintain the acidic microenvironment of the vagina, inhibiting the colonization of harmful bacteria from the source.

[0015] The acanthopanax senticosus extract contains acanthopanax glycosides and acanthopanax polysaccharides. Acanthopanax glycosides activate mucosal immune cells, promote the secretion of antimicrobial peptides, and strengthen the local defense barrier; acanthopanax polysaccharides cooperate with the microcapsule wall material to enhance the adhesion and retention ability of plant components on the vaginal mucosa; lignans such as sesamin scavenge excessive free radicals, reduce oxidative damage caused by inflammation, and delay the aging of mucosal cells.

[0016] The motherwort extract mainly contains alkaloids such as leonurine and stachydrine, and has the effects of promoting blood circulation to regulate menstruation, anti - inflammation and analgesia. It promotes blood circulation in the vaginal mucosa, accelerates the repair of damaged tissues, reduces the infiltration of inflammatory cells, and has a significant improvement effect on cervicitis and abnormal leucorrhea. In addition, motherwort can also regulate the vaginal pH value and inhibit the overgrowth of pathogenic bacteria.

[0017] The witch hazel extract has tannins and flavonoids as the main active ingredients, with the effects of astringency, bacteriostasis and barrier reinforcement. Tannins combine with mucosal proteins to form a protective film, constrict capillaries, and reduce abnormal secretions and bleeding; gallotannic acid destroys the cell membrane of anaerobic bacteria and inhibits its adhesion and colonization; flavonoid components enhance the density of the mucosal glycoprotein layer and build a physical barrier to prevent the invasion of pathogenic bacteria.

[0018] Witch hazel extract contains tannic acid and flavonoids, and has astringent, anti-inflammatory and antioxidant properties. It can constrict blood vessels, reduce vaginal mucosal bleeding, and relieve itching and burning sensations by inhibiting histamine release. Its antioxidant effect can also protect vaginal epithelial cells from free radical damage and delay mucosal aging.

[0019] Zedoary essential oil is rich in terpenoids such as germacrone and curcumol, and has broad-spectrum antibacterial and antiviral activities. Germacrone can cause the death of pathogens by destroying the sterol structure of the bacterial cell membrane and increasing membrane permeability. In addition, it also has an inhibitory effect on HPV virus and can reduce the risk of cervical lesions. The anti-inflammatory effect of zedoary essential oil can also reduce the exudate of cervical erosion tissue 10.

[0020] Jojoba oil is a natural liquid wax ester, highly compatible with human sebum, and has excellent moisturizing and repair functions. It can form a breathable protective film, reduce vaginal dryness, promote the repair of the mucosal barrier, and at the same time enhance the permeability of other active ingredients. Its mild texture can also relieve the discomfort after using the gel.

[0021] Citronella oil contains volatile components such as citronellal and geraniol, and has strong antibacterial and insecticidal effects. It inhibits the growth of pathogenic microorganisms such as Candida albicans by destroying the integrity of the fungal cell membrane, and has no obvious effect on vaginal probiotics. The cooling sensation of citronella oil can also relieve vaginal burning and odor problems.

[0022] Further, the antibacterial gel is prepared by the following steps: (1) Weigh carbomer and glycerol according to the formula amount, grind the two for 15 - 20 min to form a uniform paste. Divide the purified water into two equal parts, add half of the purified water to the paste, control the temperature at 4 - 8 °C, stir at 200 - 300 rpm for 30 min to preliminarily hydrate and disperse the carbomer to obtain a pre-swollen carbomer solution; (2) Divide triethanolamine into three equal parts. Add the first part of triethanolamine to the pre-swollen carbomer solution, stir at 400 rpm for 10 min, add a pH regulator to adjust the pH to 5.0 - 5.5; continue stirring for 20 min, add the second part of triethanolamine, add a pH regulator to adjust the pH to 4.5 - 4.8, then continue stirring for 10 min, add the third part of triethanolamine, and add a pH regulator to adjust the pH to 4.2 - 4.5; (3) Add the plant extract microcapsules to the remaining purified water, ultrasonically treat for 15 min to form a uniform suspension. Slowly add the suspension to the carbomer gel matrix obtained in step (2), homogenize and stir at 600 - 800 rpm for 30 min, then shear at 10000 rpm with a high-shear emulsifier for 5 min. After that, transfer it to a vacuum degassing tank with a vacuum degree of -0.1 MPa for degassing for 20 min to eliminate air bubbles, and fill it into an aluminum tube or a single-dose vaginal applicator under sterile conditions.

[0023] Further, the pH regulator is selected from one of tartaric acid, citric acid, potassium citrate, sodium citrate, malic acid, lactic acid, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

[0024] The beneficial effects of the present invention are as follows: (1) Through the synergistic effect of methacrylated silk fibroin and caffeic acid hyaluronic acid graft, the temperature responsiveness of the microcapsule wall material is realized, ensuring the formation of a dense network at vaginal temperature. There is a specific hydrophobic interaction between the amino acid sequence of silk fibroin and the vaginal mucosal glycoprotein. The phenolic hydroxyl group of caffeic acid is protonated under acidic conditions to enhance the charge interaction, thereby improving the adhesion of the microcapsule to the vaginal mucosal surface. The hyaluronic acid graft can be specifically recognized and hydrolyzed by the hyaluronidase secreted by vaginal lactobacilli, thereby realizing the sustained release of plant extracts. These multi-responsive characteristics enable the microcapsule to dynamically adjust its structure in the vaginal environment, and the residence time is extended compared to traditional microcapsule carriers; (2) Through the specific chelation of the o-dihydroxybenzene group of caffeic acid with silver ions, the aggregation of silver ions is prevented by steric hindrance effect, and the effective dispersion of silver ions on the surface of the microcapsule is realized. After in-situ reduction, a uniform-sized monodispersed layer of silver nanoparticles is formed on the surface of the microcapsule, effectively preventing the aggregation of silver nanoparticles. This structure greatly increases the specific surface area of silver nanoparticles compared to the traditional loading method, improves the antibacterial activity, and can effectively avoid vaginal mucosal irritation; (3) Germacrone in curcuma zedoary essential oil can insert into the sterol layer of the microbial cell membrane, increase the membrane fluidity and form pores, destroying the bacterial cell membrane. Silver nanoparticles can interfere with the electron transport chain of the bacteria and simultaneously generate reactive oxygen species to trigger lipid peroxidation, forming a synergistic antibacterial mechanism. Extracts such as bearberry leaves and motherwort are rich in flavonoids, which can promote the proliferation of lactobacilli, maintain the vaginal acidic environment, and inhibit the overgrowth of harmful bacteria; (4) When preparing the antibacterial gel of the present invention, through pre-grinding with glycerol, the intermolecular association of carbomer is destroyed by hydrogen bonds, solving the problem of low traditional swelling efficiency of carbomer, thereby facilitating the entry of plant extract microcapsules and water molecules into the interior of carbomer. Triethanolamine is added in three portions to gradually neutralize the carboxyl groups of carbomer, avoiding uneven entanglement of molecular chains caused by local over-alkalinity and forming a gradient cross-linked network to solve the problem of uneven pH distribution. The antibacterial gel prepared by the present invention has a long storage time and can be stably stored for a long time at 25°C / 60%RH. Description of the Drawings

[0025] Figure 1 It is the test result of the mucosal adhesiveness experiment of the gel prepared by the present invention; Figure 2 It is the test result of the storage stability experiment of the gel prepared by the present invention.

[0026] The accompanying drawings are used to provide a further understanding of the present invention and form a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. Detailed Description of the Invention

[0027] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred methods and materials described herein are for illustrative purposes only and do not limit the content of this application.

[0029] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0030] Example 1. A bacteriostatic gel for gynecological use containing carbomer component, comprising the following components in mass percentage: Plant extract microcapsule 0.5%, carbomer 0.5%, glycerol 2%, triethanolamine 0.1%, and the rest is purified water.

[0031] The preparation process of the plant extract microcapsule is as follows: S1. Boil silk through a 0.5% mass fraction sodium carbonate solution for 1 h to obtain degummed silk. After drying, dissolve it in a 10 mol / L lithium bromide solution, then add glycidyl methacrylate, and react at 60 °C for 4 h under nitrogen protection. Dialyze the reaction solution with a filter membrane with a molecular weight cut-off of 8 kDa, and freeze-dry to obtain methacrylated silk fibroin; The mass ratio of the degummed silk to the lithium bromide solution is 1:10, and the dosage of glycidyl methacrylate is 10% of the mass of silk fibroin; S2. Dissolve hyaluronic acid in MES buffer with pH = 5.0, add EDC and NHS, stir in the dark at room temperature for 2 h to form an active hyaluronic acid solution; dissolve caffeic acid in dimethyl sulfoxide, add HOBt and DIC, stir at room temperature for 1 h to obtain an activated caffeic acid solution, mix the active hyaluronic acid solution with the activated caffeic acid solution, adjust the pH to 7.4, react in the dark at 50 °C for 24 h, filter the reaction solution through a 0.45 μm filter membrane, dialyze with a filter membrane with a molecular weight cut-off of 3.5 kDa, and freeze-dry to obtain a caffeic acid-hyaluronic acid graft; The mass of the MES buffer is 20 times that of hyaluronic acid; the molar amount of EDC is 1.5 times that of the carboxyl group of hyaluronic acid, and the molar amount of NHS is 0.5 times that of EDC; the mass of dimethyl sulfoxide is 20 times that of caffeic acid, and the molar amounts of HOBt, DIC and caffeic acid are 1.0:1.5:1; the mass ratio of the active hyaluronic acid solution to the activated caffeic acid solution is 1:1.2; S3. Mix methacrylated silk fibroin with the caffeic acid-hyaluronic acid graft and add them to an ethanol solution, add tartaric acid to adjust the pH to 5.0 to form a homogeneous solution. After mixing the composite plant extract with Tween 80, slowly add it to the homogeneous solution. Drop an aqueous silver nitrate solution with a mass concentration of 0.01% into the homogeneous solution, then add sodium sulfite, react in the dark at 50 °C for 30 min, then add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate accounting for 0.25% of the mass of the homogeneous solution, cure by ultraviolet light, then centrifuge at 4000 rpm for 10 min to collect the microcapsules, wash 3 times with deionized water, and freeze-dry to obtain plant extract microcapsules loaded with silver nanoparticles; The mass ratio of methacrylated silk fibroin to the caffeic acid-hyaluronic acid graft is 1:2; in step S3, the mass of the ethanol solution is 10 times that of methacrylated silk fibroin and the caffeic acid-hyaluronic acid graft, and the mass fraction of the ethanol solution is 60%; the mass ratio of the composite plant extract to Tween 80 is 10:1; the mass of the aqueous silver nitrate solution is 5% of the homogeneous solution, and the mass of sodium sulfite is 0.02% of the homogeneous solution.

[0032] The composite plant extract includes the following components in parts by mass: 0.5 part of bearberry leaf extract, acanthopanax extract, 1 part of motherwort extract, 1 part of witch hazel extract, 25 parts of zedoary essential oil, 3 parts of jojoba oil, and 2 parts of citronella oil.

[0033] The antibacterial gel is prepared by the following steps: (1)Weigh carbomer and glycerol according to the formula amount, grind the two for 15 min to form a uniform paste. Divide the purified water into two equal parts, add half of the purified water to the paste, control the temperature at 4 °C, stir at 200 rpm for 30 min to preliminarily hydrate and disperse the carbomer to obtain a pre-swollen carbomer solution; (2)Divide triethanolamine into three equal parts. Add the first part of triethanolamine to the pre-swollen carbomer solution, stir at 400 rpm for 10 min, add tartaric acid to adjust the pH to 5.0; continue stirring for 20 min, add the second part of triethanolamine, add tartaric acid to adjust the pH to 4.5, then continue stirring for 10 min, add the third part of triethanolamine, and add tartaric acid to adjust the pH to 4.2; (3)Add the plant extract microcapsules to the remaining purified water, ultrasonically treat for 15 min to form a uniform suspension. Slowly add the suspension to the carbomer gel matrix obtained in step (2), homogenize and stir at 600 rpm for 30 min, then shear at 10000 rpm with a high-shear emulsifier for 5 min. After that, transfer it to a vacuum degassing tank with a vacuum degree of -0.1 MPa for degassing for 20 min to eliminate air bubbles, and fill it into an aluminum tube or a single-dose vaginal applicator under sterile conditions.

[0034] Example 2. A bacteriostatic gel for gynecological use containing carbomer components, comprising the following components in mass percentage: Plant extract microcapsules 5%, carbomer 3%, glycerol 10%, triethanolamine 1.5%, and the rest is purified water.

[0035] The preparation process of the plant extract microcapsules is as follows: S1. Boil silk in a 0.5% mass fraction sodium carbonate solution for 1 h to obtain degummed silk. After drying, dissolve it in a 10 mol / L lithium bromide solution, then add glycidyl methacrylate, and react at 60 °C for 4 h under nitrogen protection. Dialyze the reaction solution with a filter membrane with a molecular weight cut-off of 8 kDa, and freeze-dry to obtain methacrylated silk fibroin; The mass ratio of the degummed silk to the lithium bromide solution is 1:15, and the dosage of glycidyl methacrylate is 20% of the mass of silk fibroin; S2. Dissolve hyaluronic acid in a MES buffer solution with pH = 5.0, add EDC and NHS, stir at room temperature in the dark for 2 h to form an active hyaluronic acid solution; dissolve caffeic acid in dimethyl sulfoxide, add HOBt and DIC, stir at room temperature for 1 h to obtain an activated caffeic acid solution. Mix the active hyaluronic acid solution and the activated caffeic acid solution, adjust the pH to 7.4, and react at 50 °C in the dark for 24 h. Filter the reaction solution through a 0.45 μm filter membrane, dialyze it with a filter membrane with a molecular weight cut-off of 3.5 kDa, and freeze-dry to obtain a caffeic acid-hyaluronic acid graft; The mass of the MES buffer solution is 30 times that of hyaluronic acid; the molar amount of EDC is 1.8 times that of the carboxyl group of hyaluronic acid, and the molar amount of NHS is 0.8 times that of EDC; the mass of dimethyl sulfoxide is 30 times that of caffeic acid, and the molar amounts of HOBt, DIC and caffeic acid are 1.2:1.8:1; the mass ratio of the hyaluronic acid active solution to the caffeic acid activation solution is 1:1.5; S3. Mix methacrylated silk fibroin and caffeic acid hyaluronic acid graft and add them to an ethanol solution. Add tartaric acid to adjust the pH to 5.5 to form a homogeneous solution. After mixing the composite plant extract and Tween 80, slowly add them to the homogeneous solution. Drop an aqueous silver nitrate solution with a mass concentration of 0.01% into the homogeneous solution, then add sodium sulfite, react at 50 °C in the dark for 30 min, then add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate accounting for 0.25% of the mass of the homogeneous solution, cure by ultraviolet light, then centrifuge at 4000 rpm for 10 min to collect microcapsules, wash them 3 times with deionized water, and freeze-dry to obtain plant extract microcapsules loaded with silver nanoparticles; The mass ratio of methacrylated silk fibroin to caffeic acid hyaluronic acid graft is 1:2; in step S3, the mass of the ethanol solution is 15 times that of methacrylated silk fibroin and caffeic acid hyaluronic acid graft, and the mass fraction of the ethanol solution is 75%; the mass ratio of the composite plant extract to Tween 80 is 15:1; the mass of the aqueous silver nitrate solution is 10% of the homogeneous solution, and the mass of sodium sulfite is 0.05% of the homogeneous solution.

[0036] The composite plant extract includes the following components in parts by mass: 2 parts of bearberry leaf extract, acanthopanax extract, 3 parts of motherwort extract, 3 parts of witch hazel extract, 30 parts of zedoary essential oil, 5 parts of jojoba oil, and 4 parts of citronella oil.

[0037] The antibacterial gel is prepared by the following steps: (1) Weigh carbomer and glycerol according to the formula amount, grind them for 20 min to form a uniform paste. Divide the purified water into two equal parts, add half of the purified water to the paste, control the temperature at 8 °C, and stir at 300 rpm for 30 min to preliminarily hydrate and disperse the carbomer to obtain a pre-swollen carbomer solution; (2) Divide triethanolamine into three equal parts. Add the first part of triethanolamine to the pre-swollen carbomer solution, stir at 400 rpm for 10 min, add citric acid to adjust the pH to 5.5; continue to stir for 20 min and add the second part of triethanolamine, add citric acid to adjust the pH to 4.8, then continue to stir for 10 min and add the third part of triethanolamine, add citric acid to adjust the pH to 4.5; (3) Add the plant extract microcapsules to the remaining purified water, and ultrasonically treat for 15 min to form a uniform suspension. Slowly add the suspension to the carbomer gel matrix obtained in step (2), and homogenize and stir at 800 rpm for 30 min. Then, shear at 10,000 rpm using a high-shear emulsifier for 5 min. After that, transfer it to a vacuum degassing tank with a vacuum degree of -0.1 MPa for 20 min to remove air bubbles. Fill it into an aluminum tube or a single-dose vaginal applicator under aseptic conditions.

[0038] Example 3. A bacteriostatic gel for gynecological use containing carbomer components, comprising the following components in mass percentage: Plant extract microcapsules 2.75%, carbomer 1.75%, glycerol 6%, triethanolamine 0.8%, and the rest is purified water.

[0039] The preparation process of the plant extract microcapsules is as follows: S1. Boil silk in a 0.5% mass fraction sodium carbonate solution for 1 h to obtain degummed silk. After drying, dissolve it in a 10 mol / L lithium bromide solution, then add glycidyl methacrylate, and react at 60 °C for 4 h under nitrogen protection. Dialyze the reaction solution using a filter membrane with a molecular weight cut-off of 8 kDa, and freeze-dry to obtain methacrylated silk fibroin. The mass ratio of the degummed silk to the lithium bromide solution is 1:12.5, and the dosage of glycidyl methacrylate is 15% of the mass of silk fibroin. S2. Dissolve hyaluronic acid in an MES buffer solution with pH = 5.0, add EDC and NHS, and stir at room temperature in the dark for 2 h to form an active hyaluronic acid solution; dissolve caffeic acid in dimethyl sulfoxide, add HOBt and DIC, and stir at room temperature for 1 h to obtain an activated caffeic acid solution. Mix the active hyaluronic acid solution and the activated caffeic acid solution, adjust the pH to 7.4, and react at 50 °C in the dark for 24 h. Filter the reaction solution through a 0.45 μm filter membrane, dialyze it using a filter membrane with a molecular weight cut-off of 3.5 kDa, and freeze-dry to obtain a caffeic acid-hyaluronic acid graft. The mass of the MES buffer solution is 25 times that of hyaluronic acid; the molar amount of EDC is 1.65 times that of the carboxyl group of hyaluronic acid, and the molar amount of NHS is 0.65 times that of EDC; the mass of dimethyl sulfoxide is 25 times that of caffeic acid, and the molar amounts of HOBt, DIC, and caffeic acid are 1.1:1.65:1; the mass ratio of the active hyaluronic acid solution to the activated caffeic acid solution is 1:1.35.

[0040] S3. Mix methacrylated silk fibroin with caffeic acid hyaluronic acid graft, add the mixture to an ethanol solution, add tartaric acid to adjust the pH to 5.25 to form a homogeneous solution. After mixing the composite plant extract with Tween 80, slowly add it to the homogeneous solution. Drop an aqueous silver nitrate solution with a mass concentration of 0.01% into the homogeneous solution, then add sodium sulfite, and react at 50 °C in the dark for 30 min. Then add lithium phenyl(2,4,6-trimethylbenzoyl)phosphate accounting for 0.25% of the mass of the homogeneous solution, cure it by ultraviolet light, then centrifuge at 4000 rpm for 10 min to collect the microcapsules, wash them 3 times with deionized water, and freeze-dry to obtain plant extract microcapsules loaded with silver nanoparticles; the mass ratio of methacrylated silk fibroin to caffeic acid hyaluronic acid graft is 1:2; in step S3, the mass of the ethanol solution is 12.5 times that of methacrylated silk fibroin and caffeic acid hyaluronic acid graft, and the mass fraction of the ethanol solution is 67.5%; the mass ratio of the composite plant extract to Tween 80 is 12.5:1; the mass of the aqueous silver nitrate solution is 7.5% of the homogeneous solution, and the mass of sodium sulfite is 0.03% of the homogeneous solution.

[0041] The composite plant extract comprises the following components in parts by mass: 1.25 parts of bearberry leaf extract, acanthopanax extract, 2 parts of motherwort extract, 2 parts of witch hazel extract, 27.5 parts of zedoary essential oil, 4 parts of jojoba oil, and 3 parts of citronella oil.

[0042] The antibacterial gel is prepared by the following steps: (1) Weigh carbomer and glycerol according to the formula amount, grind the two for 17.5 min to form a uniform paste. Divide the purified water into two equal parts, add half of the purified water to the paste, control the temperature at 6 °C, and stir at 250 rpm for 30 min to preliminarily hydrate and disperse the carbomer to obtain a pre-swollen carbomer solution. (2) Divide triethanolamine into three equal parts. Add the first part of triethanolamine to the pre-swollen carbomer solution, stir at 400 rpm for 10 min, add potassium dihydrogen phosphate to adjust the pH to 5.0; continue stirring for 20 min, add the second part of triethanolamine, add potassium dihydrogen phosphate to adjust the pH to 4.6, then continue stirring for 10 min, add the third part of triethanolamine, and add potassium dihydrogen phosphate to adjust the pH to 4.3. (3) Add the plant extract microcapsules to the remaining purified water, ultrasonically treat for 15 min to form a uniform suspension, slowly add the suspension to the carbomer gel matrix obtained in step (2), homogenize and stir at 700 rpm for 30 min, then shear at 10000 rpm with a high-shear emulsifier for 5 min, and then transfer it to a vacuum degassing tank with a vacuum degree of -0.1 MPa for degassing for 20 min to remove bubbles, and fill it into an aluminum tube or a single-dose vaginal applicator under sterile conditions.

[0043] Comparative Example 1. In this comparative example, the microcapsule preparation step was omitted, and the composite plant extract was directly mixed with Tween 80 and then added to the gel matrix. The others were the same as in Example 3.

[0044] Comparative Example 2. In this comparative example, the steps of silver nitrate and sodium sulfite were omitted during microcapsule preparation. The others were the same as in Example 3.

[0045] Comparative Example 3. In this comparative example, gelatin and gum arabic were used to coacervate and encapsulate the plant extract at a mass ratio of 1:1. The total mass of gelatin and gum arabic was the same as that of methacrylated silk fibroin and hyaluronic acid grafted with caffeic acid. The others were the same as in Example 3.

[0046] The specific preparation steps are as follows: Gelatin and gum arabic were mixed and added to an ethanol solution. Tartaric acid was added to adjust the pH to 5.25 to form a homogeneous solution. After the composite plant extract was mixed with Tween 80, it was slowly added to the homogeneous solution. An aqueous silver nitrate solution with a mass concentration of 0.01% was added dropwise to the homogeneous solution, and then sodium sulfite was added. The reaction was carried out at 50 °C in the dark for 30 min, and then the microcapsules were collected by centrifugation at 4000 rpm for 10 min, washed 3 times with deionized water, and freeze-dried to obtain plant extract microcapsules loaded with silver nanoparticles.

[0047] Comparative Example 4. In this comparative example, triethanolamine was added to the carbomer slurry at one time, and then the pH was adjusted to 4.2 - 4.5. The others were the same as in Example 3.

[0048] Result analysis Test Example 1. Irritation experiment Human vaginal epithelial cells (VK2 / E6E7) were cultured to the logarithmic growth phase, digested with 0.25% trypsin, and the cell concentration was adjusted to 5×10 4 cells / mL. 100 μL of the cell suspension was added to each well of a 96-well plate and cultured in an incubator at 37 °C and 5% CO 2 for 24 h until the cell attachment rate reached 80% - 90%.

[0049] 0.5 g of the gel samples of Examples 1 - 3 and Comparative Examples 1 - 4 were added with 5 mL of PBS (pH 7.4), and extracted by constant temperature oscillation at 37 °C for 24 h to obtain the initial extract (concentration 100 mg / mL). Then the initial extract was serially diluted with PBS to 10 mg / mL, 1 mg / mL, and 0.1 mg / mL as the experimental extracts.

[0050] Discard the old culture medium in the 96-well plate, and add 100 μL of gel extracts with different concentrations to each well. Set up a blank control group (add 100 μL of complete culture medium) and a positive control group (add culture medium containing 0.1% Triton X-100).

[0051] After continuous culture for 24 h, add 20 μL of MTT reagent to each well and incubate in the dark at 37 °C for 4 h.

[0052] Discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 min to dissolve the formazan crystals.

[0053] Measure the OD 490 value of each well with an enzyme-linked immunosorbent assay (ELISA) reader and calculate the cell survival rate.

[0054] The above test results are shown in the following table.

[0055] Table 1 Test results of gel irritation experiment Sample 0.1 mg / mL (%) 1 mg / mL (%) 10 mg / mL (%) Example 1 98.2±1.1 97.5±0.8 96.2±1.3 Example 2 97.8±0.9 96.7±1.2 95.4±1.0 Example 3 99.1±0.7 98.3±0.8 97.2±0.9 Comparative Example 1 89.5±1.5 85.2±2.1 78.6±1.5 Comparative Example 2 97.5±0.6 97.2±0.4 95.9±0.7 Comparative Example 3 93.2±1.2 90.1±1.7 87.4±1.4 Comparative Example 4 82.6±1.8 79.3±2.0 76.6±1.8 As can be seen from the results in Table 1, the cell survival rates of Examples 1-3 are all ≥ 95%, indicating that the gel has no obvious irritation to vaginal epithelial cells and meets the safety requirements for gynecological drugs. In Comparative Example 1, the microcapsule preparation step was omitted, and the survival rate decreased significantly, indicating that when the plant extract directly contacts the cells, cell damage may be caused by too high component concentration or excipient irritation, and the encapsulation effect of the microcapsule can reduce irritation. The survival rate of Comparative Example 4 is the lowest because the local over-alkaline environment destroys the uniformity of the carbomer molecular chain, resulting in a sudden pH change that stimulates the cells, verifying the necessity of adjusting the pH in stages. Comparative Example 3 uses the traditional gelatin - gum arabic wall material, and the survival rate is lower than that of the examples, indicating that the methacrylated silk fibroin - caffeic acid hyaluronic acid graft wall material of the present invention is milder and has better biocompatibility.

[0056] Test Example 2. Antibacterial experiment Use Candida albicans (ATCC 10231), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC6538), and Gardnerella vaginalis (ATCC 14018) as test strains. Inoculate each strain into the corresponding culture medium and culture at 37 °C (28 °C for fungi) for 24 h. Adjust the bacterial liquid concentration to 1×10 8 CFU / mL with sterile normal saline. Take 100 μL of the bacterial liquid and evenly coat it on the surface of the corresponding agar plate, and let it stand for 5 min until the bacterial liquid is absorbed. Place a sterile Oxford cup on the plate, and add 50 μL of the gel samples of Examples 1-3 and Comparative Examples 1-4 to each cup. The control group adds an equal amount of sterile normal saline. Incubate the bacterial plates at 37 °C for 24 h and the fungal plates at 28 °C for 48 h. Measure the diameter of the inhibition zone with a vernier caliper. The above test results are shown in the following table.

[0057] Table 2 Test Results of Gel Bacteriostasis Experiment Sample Candida albicans Escherichia coli Staphylococcus aureus Gardnerella Example 1 18.5±0.3 22.1±0.5 24.3±0.4 19.8±0.6 Example 2 19.2±0.4 23.5±0.6 25.1±0.3 20.5±0.5 Example 3 21.4±0.5 25.3±0.4 26.8±0.6 21.1±0.8 Comparative Example 1 12.3±0.6 15.4±0.7 16.9±0.5 13.8±0.9 Comparative Example 2 14.1±0.4 16.2±0.5 17.5±0.6 15.1±0.8 Comparative Example 3 16.8±0.5 19.3±0.6 20.7±0.4 17.6±0.7 Comparative Example 4 17.9±0.7 21.2±0.8 22.1±0.5 18.6±1.0 As can be seen from the results in Table 1, the gels of each example showed significant bacteriostatic effects on 4 kinds of pathogenic bacteria. The diameter of the bacteriostatic zone was significantly larger than that of the comparative examples. In Comparative Example 1, the plant extract was not encapsulated, the nano-silver was prone to agglomeration, the dispersibility of the effective antibacterial components was poor, and the bacteriostatic activity decreased. In Comparative Example 2, the synergistic antibacterial effect of nano-silver and zedoary essential oil was lacking, and the bacteriostatic effect decreased. In Comparative Example 3, the traditional wall material was used and it was impossible to achieve the monodisperse loading of nano-silver and the slow release of the plant extract, and the release of the antibacterial components was uneven, and the effect weakened. In Comparative Example 4, the viscosity of the carbomer matrix was uneven, which affected the drug release rate, and the bacteriostatic zone was also slightly smaller than that of the examples.

[0058] Test Example 3. Mucosal Adhesion Experiment Add 0.1% sodium fluorescein (w / v) to the gel samples of Examples 1-3 and Comparative Examples 1-4, disperse them evenly by ultrasonic wave to prepare fluorescently labeled gels. After anesthetizing New Zealand white rabbits intraperitoneally with sodium pentobarbital (30 mg / kg), slowly inject 0.5 g of the fluorescent gel into the deep vagina with a sterile single-dose vaginal applicator, avoiding outflow. At 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48 h after administration, irrigate the vagina with 5 mL of sterile normal saline, and collect the irrigation fluid. Centrifuge the irrigation fluid at 3000 rpm for 10 min, take the supernatant, and measure the fluorescence intensity with a fluorescence spectrometer (excitation wavelength 490 nm, emission wavelength 520 nm). With time as the abscissa and fluorescence intensity as the ordinate, draw a retention curve and calculate the retention time of the gel in the vagina (the time when the fluorescence intensity drops to 50% of the initial value). The above test results are shown in Figure 1 。

[0059] From Figure 1 the data, it can be seen that the vaginal retention time (the time when the fluorescence intensity drops to 50%) of the gels of the examples was all > 24 h, which could better prolong the action time of the active substances of the plant extract, so as to give full play to the curative effect.

[0060] Test Example 4. Storage Stability Experiment Store the gel samples of Examples 1-3 and Comparative Examples 1-4 at 25 °C / 60% RH for 6 months, use a rheometer to measure the viscosity of the gels before and after storage, and calculate the gel viscosity reduction rate. The above test results are shown in Figure 2 。

[0061] From Figure 1The data shows that after the gel of the embodiment is stored at 25°C / 60%RH for 6 months, the viscosity reduction rate is less than 10%; the viscosity reduction rate of comparative example 4 is greater than 30%, and the reduction rate of other comparative examples is 15%-25%. The embodiment is pre-grinded with glycerol, and the carbomer carboxyl groups are neutralized in stages to form a gradient cross-linked network, avoid local over-alkalinity leading to entanglement of molecular chains, and have a uniform pH distribution to adapt to the acidic environment of the vagina and maintain stable viscosity. Comparative example 4 adds triethanolamine at one time. Due to the excessive local alkalinity, the carbomer molecular chains are over-cross-linked, the structure is loose during storage, and the viscosity drops sharply, so the long-term storage stability is poor.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0063] The present invention and its implementation methods are described above, which is not restrictive. The drawings are only one of the implementation methods of the present invention, and the actual application is not limited thereto. In short, if ordinary technicians in the field are inspired by it and design methods and embodiments similar to the technical solution without creativity without departing from the purpose of the invention, they should all fall within the protection scope of the present invention.

Claims

1. A gynecological antibacterial gel containing carbomer, characterized in that: The invention comprises the following components in percentage by weight: 0.5-5% of plant extract microcapsules, 0.5-3% of carbomer, 2-10% of glycerol, 0.1-1.5% of triethanolamine, and the rest of purified water; The preparation process of the plant extract microcapsules is as follows: S1. The silk was boiled in a 0.5% mass fraction sodium carbonate solution for 1 hour to obtain degummed silk, which was then dried and dissolved in a 10 mol / L lithium bromide solution. Glycidyl methacrylate was then added and reacted at 60°C for 4 hours under nitrogen protection. The reaction solution was dialyzed using a filter membrane with a molecular weight cutoff of 8 kDa, and methacrylylated silk fibroin was obtained after freeze-drying. S2. Dissolve hyaluronic acid in MES buffer at pH=5.0, add EDC and NHS, stir at room temperature in the dark for 2 hours to form a hyaluronic acid active solution; dissolve caffeic acid in dimethyl sulfoxide, add HOBt and DIC, stir at room temperature for 1 hour to obtain a caffeic acid activated solution, mix the hyaluronic acid active solution with the caffeic acid activated solution, adjust the pH to 7.4, react at 50°C in the dark for 24 hours, filter the reaction solution through a 0.45μm filter membrane, dialyze with a filter membrane with a molecular weight cutoff of 3.5kDa, and freeze-dry to obtain a caffeic acid hyaluronic acid graft; S3. Mix methacrylylated silk protein and caffeic acid hyaluronic acid grafts and add them to an ethanol solution, add tartaric acid to adjust the pH to 5.0-5.5 to form a homogeneous solution, mix the composite plant extract with Tween 80, and slowly add the mixture to the homogeneous solution, drop a 0.01% silver nitrate aqueous solution into the homogeneous solution, add sodium sulfite, react at 50°C in the dark for 30 minutes, add 0.25% of the mass of the homogeneous solution of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, treat with ultraviolet light at a wavelength of 365-400nm for 100-120s, then collect the microcapsules by centrifugation at 4000rpm for 10min, wash three times with deionized water, and freeze-dry to obtain plant extract microcapsules loaded with nanosilver.

2. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass ratio of the degummed silk to the lithium bromide solution is 1:10-15, and the amount of glycidyl methacrylate is 10-20% of the mass of silk fibroin.

3. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass of the MES buffer is 20-30 times that of hyaluronic acid; the molar amount of the EDC is 1.5-1.8 times that of the carboxyl group of hyaluronic acid, and the molar amount of the NHS is 0.5-0.8 times that of EDC; the mass of the dimethyl sulfoxide is 20-30 times that of caffeic acid, and the molar amounts of the HOBt, DIC and caffeic acid are 1.0-1.2:1.5-1.8:

1.

4. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass ratio of the hyaluronic acid active solution to the caffeic acid activation solution is 1:1.2-1.

5.

5. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass ratio of the methacrylylated silk fibroin to the caffeic acid hyaluronic acid graft is 1:

2.

6. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass of the ethanol solution in step S3 is 10-15 times that of the methacrylylated silk fibroin and caffeic acid hyaluronic acid graft, and the mass fraction of the ethanol solution is 60-75%.

7. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The mass ratio of the composite plant extract to Tween 80 is 10-15:1; the mass of the silver nitrate aqueous solution is 5-10% of the homogeneous solution, and the mass of the sodium sulfite is 0.02-0.05% of the homogeneous solution.

8. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The composite plant extract comprises the following components in parts by weight: 0.5-2 parts of bearberry leaf extract, 1-3 parts of Acanthopanax senticosus extract, 1-3 parts of Leonurus japonicus extract, 1-3 parts of Hamamelis virginiana extract, 25-30 parts of Curcuma zedoaria essential oil, 3-5 parts of jojoba oil and 2-4 parts of citronella oil.

9. The gynecological antibacterial gel containing carbomer ingredients according to claim 1, characterized in that: The antibacterial gel is prepared by the following steps: (1) Weigh carbomer and glycerin according to the formula amount, grind the two for 15-20 minutes to form a uniform slurry, divide the purified water into two equal parts, add half of the purified water to the slurry, control the temperature at 4-8°C, and stir at 200-300 rpm for 30 minutes to allow the carbomer to be initially hydrated and dispersed to obtain a pre-swollen carbomer solution; (2) Divide triethanolamine into three equal parts, add the first part of triethanolamine to the pre-swollen carbomer solution, stir at 400 rpm for 10 minutes, add a pH adjuster to adjust the pH to 5.0-5.5; continue stirring for 20 minutes, add the second part of triethanolamine, add a pH adjuster to adjust the pH to 4.5-4.8, continue stirring for 10 minutes, add the third part of triethanolamine, and add a pH adjuster to adjust the pH to 4.2-4.5; (3) Add the plant extract microcapsules to the remaining purified water and perform ultrasonic treatment for 15 minutes to form a uniform suspension. Slowly add the suspension to the carbomer gel matrix obtained in step (2), homogenize and stir at 600-800 rpm for 30 minutes, and then use a high shear emulsifier to shear at 10,000 rpm for 5 minutes. Then transfer to a vacuum degassing tank with a vacuum degree of -0.1 MPa for degassing for 20 minutes to eliminate bubbles, and fill into an aluminum tube or a single-dose vaginal applicator under sterile conditions.

10. The gynecological antibacterial gel containing carbomer ingredients according to claim 9, characterized in that: The pH regulator is selected from one of tartaric acid, citric acid, potassium citrate, sodium citrate, malic acid, lactic acid, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.

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