Medicine for repairing oral mucosa and preparation method thereof

Abstract

The invention relates to an oral mucosa repairing medicine and a preparation method thereof, and belongs to the technical field of medicines, the medicine comprises barnacle peptide, salivary lactobacillus, resveratrol, a biphasic adhesion matrix, hydroxyapatite, vitamin B family and other components. The preparation method comprises the following steps: sequentially carrying out enzymolysis on barnacle gooseneck by pepsin and lumbrukinase to obtain barnacle peptide. The double-phase adhesion matrix comprises a water-phase matrix, an oil-phase matrix and lecithin; the water-phase matrix contains a chondroitin sulfate-chitosan quaternary ammonium salt compound, sodium alginate and other components; the oil-phase matrix comprises sesame oil, beeswax, a polylactic acid-glycolic acid copolymer and the like. According to the invention, barnacle peptide and salivary lactobacillus are prepared by adopting a special method and are matched with resveratrol for use, so that multi-target and multi-path promotion of oral mucosa repair can be realized; the chondroitin sulfate-chitosan quaternary ammonium salt compound is prepared through electrostatic and physical embedding, the chondroitin sulfate-chitosan quaternary ammonium salt compound is matched with other components to prepare the biphasic adhesion matrix, the biocompatibility is good, the adhesion is high, and the action time of a sustained-release drug is well prolonged.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a medicine for repairing oral mucosa and a preparation method thereof. Background Art

[0002] The oral mucosa serves as a vital barrier against external irritants, playing a critical role in maintaining oral health and normal physiological function. However, various factors can lead to damage to the oral mucosa, causing ulcers, inflammation, and other conditions. These conditions not only cause discomfort such as pain and difficulty eating, but can also affect communication and quality of life. In severe cases, they can even lead to the spread of infection, impacting overall health. Therefore, the research and development of oral mucosal repair drugs is of great importance. Trauma is a common cause of oral mucosal damage. Physical trauma, such as surgery, accidental bites, and burns, as well as chemical trauma, such as contact with corrosive substances, can damage the integrity of the oral mucosa. Furthermore, long-term wear of ill-fitting dentures can cause continuous friction and pressure on the oral mucosa, leading to traumatic ulcers. Infectious factors, such as invasion by pathogens like viruses, bacteria, and fungi, can also cause damage to the oral mucosa. For example, oral herpes, caused by the herpes simplex virus, and oral thrush, caused by Candida albicans, not only damage the mucosal tissue but also disrupt the normal microbial balance in the mouth, making repair more difficult. With the accelerating pace of life and increasing mental stress, immune-related diseases such as recurrent aphthous ulcers and pemphigus are becoming increasingly common among oral mucosal diseases. These diseases, caused by autoimmune system disorders, lead to recurrent ulcers and erosions in the oral mucosa. Traditional treatments are difficult to cure and are prone to recurring attacks, causing long-term pain for patients.

[0003] At present, commonly used oral mucosal repair drugs mainly include local pastes, patches, sprays and other dosage forms. Although these drugs can relieve symptoms to a certain extent, they still have many limitations. For example, traditional paste drugs have poor adhesion in the oral cavity and are easily washed away by saliva, making it difficult to maintain effective drug concentrations at the site of injury, affecting the repair effect; although patch drugs can adhere to the mucosa well, they have problems such as loose adhesion and strong local irritation; spray drugs are easy to use, but the drug is unevenly distributed on the mucosal surface and has a short duration of action. In addition, existing repair drugs lack the coordinated regulation of multiple complex mechanisms in the oral mucosal repair process, and it is difficult to meet the clinical demand for efficient and rapid repair of oral mucosa.

[0004] Therefore, the development of new oral mucosal repair drugs that overcome the shortcomings of existing drugs and have good biocompatibility and adhesion and can promote oral mucosal repair through multiple targets and multiple pathways has become an urgent problem to be solved in the current oral medicine field. Summary of the Invention

[0005] In view of the existing oral mucosa repair drugs having poor adhesiveness, being difficult to maintain an effective drug concentration at the injury site, and affecting the repair effect; the repair drugs lack the synergistic regulation of various complex mechanisms during the oral mucosa repair process and are difficult to meet the clinical demand for efficient and rapid repair of oral mucosa. The present invention provides a repair drug for oral mucosa and its preparation method. Barnacle peptide is prepared by a special enzymatic hydrolysis method, and lactocin is prepared by a special fermentation and extraction method. The combination of barnacle peptide, lactocin and resveratrol in a certain proportion can promote oral mucosa repair through multiple targets and multiple pathways; chondroitin sulfate-chitosan quaternary ammonium salt complex is prepared by electrostatic and physical embedding, and a biphasic adhesion matrix is prepared in combination with other components, which has good biocompatibility, strong adhesiveness, can well extend the sustained-release drug action time, and improve the mucosal repair performance. The specific technical solution is as follows: A repair drug for oral mucosa is made from the following raw materials in parts by mass: 2 to 5 parts of barnacle peptide, 1 to 3 parts of lactocin, 0.5 to 2 parts of resveratrol, 70 to 85 parts of biphasic adhesion matrix, 5 to 10 parts of hydroxyapatite, 0.5 to 3 parts of vitamin B group, and the balance is deionized water with a water content of 4wt% to 8wt%; The barnacle peptide contains the product between 1 kDa and 3 kDa obtained by successively enzymatically hydrolyzing the crushed barnacle soft body with pepsin and lumbrokinase; The component mass ratio of the biphasic adhesion matrix is aqueous phase matrix: oil phase matrix: lecithin = (7 - 8): (4 - 5): (0.3 - 0.5); the component mass ratio of the aqueous phase matrix is chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate = (1 - 1.5): (18 - 22): (0.5 - 0.8); the component mass ratio of the oil phase matrix is sesame oil: beeswax: poly(lactic-co-glycolic acid): dimethicone = (90 - 100): (3 - 5): (8 - 10): (0.3 - 0.6); The composite mass ratio of chondroitin sulfate and chitosan quaternary ammonium salt in the chondroitin sulfate-chitosan quaternary ammonium salt complex is (1.5 - 2.0): (0.18 - 0.25).

[0006] In the above drug, the preparation method of barnacle peptide includes: crushing the barnacle soft body to obtain a crushed product, adding hydrochloric acid aqueous solution, adding 1% - 2% of pepsin based on the mass of the crushed product, enzymatically hydrolyzing at 35°C - 40°C for 1h - 2h, adjusting the pH to 7 - 8, adding 1.5% - 3% of lumbrokinase based on the mass of the crushed product, enzymatically hydrolyzing at 35°C - 40°C for 1.5h - 2h, inactivating the enzyme, centrifuging to take the supernatant, performing ultrafiltration using an ultrafiltration membrane to obtain the retentate between 1 kDa and 3 kDa, and freeze-drying to obtain barnacle peptide.

[0007] In the above method for preparing barnacle peptides, the amount of hydrochloric acid aqueous solution is 8 to 10 times the mass of the pulverized material; the pH value of the hydrochloric acid aqueous solution is 1.5 to 2.5; enzyme inactivation is carried out at 85°C to 90°C for 10 min to 15 min; centrifugation is carried out at 4000 r / min to 5000 r / min for 10 min to 15 min.

[0008] In the above drug, the preparation method of lactobacillin includes: adding 2 wt% to 3 wt% of activated Lactobacillus salivarius, 1 wt% to 1.5 wt% of celery juice, 0.5 wt% to 1 wt% of bitter gourd juice, and 0.1 wt% to 0.2 wt% of glucose oxidase to MRS medium, anaerobically culturing at 36°C to 38°C for 48 h to 72 h to obtain a bacterial solution, centrifuging at 6000 r / min to 8000 r / min for 15 min to 20 min, collecting the supernatant, adding 50 wt% to 60 wt% of ammonium sulfate based on the mass of the supernatant, standing at 4°C to 6°C for 12 h to 18 h, precipitating the bacteriocin, centrifuging at 8000 r / min to 10000 r / min for 20 min to 30 min, collecting the precipitate, using a dialysis bag with a cut-off molecular weight of 1 kDa, dialyzing in flowing deionized water for 12 h to 24 h, and freeze-drying to obtain lactobacillin.

[0009] In the above drug, the preparation method of chondroitin sulfate-chitosan quaternary ammonium salt complex includes: by mass: dissolving 1.5 parts to 2.0 parts of chondroitin sulfate in 80 parts to 100 parts of phosphate buffer solution with a pH of 5.3 to 5.6 to form solution A; dissolving 0.18 parts to 0.25 parts of chitosan quaternary ammonium salt in 50 parts to 60 parts of phosphate buffer solution with a pH of 5.3 to 5.6, then adding 0.5 parts to 1.0 parts of polyethylene glycol and 0.3 parts to 0.5 parts of sodium chloride, stirring evenly to form solution B; under stirring, dropping solution B into solution A, after dropping, continue stirring and reacting to fully carry out electrostatic binding to obtain a reaction solution, standing at low temperature to promote the contraction and entanglement of molecular chains, physically embedding the electrostatically bound chitosan quaternary ammonium salt molecules in the chondroitin sulfate network structure; using a dialysis bag with a cut-off molecular weight of 1 kDa, dialyzing in flowing deionized water for 12 h to 24 h, and freeze-drying to obtain chondroitin sulfate-chitosan quaternary ammonium salt complex.

[0010] In the above preparation method of chondroitin sulfate-chitosan quaternary ammonium salt complex, the stirring speed is 200 r / min to 250 r / min; the dropping speed is 1 mL / min to 2 mL / min; the reaction time is 1 h to 1.5 h; low-temperature standing is carried out at 4°C to 6°C for 2 h to 3 h.

[0011] In the above drug, the B vitamins are a mixture of VB1, VB2, VB6, and VB12.

[0012] The preparation method of the above-mentioned oral mucosa repair drug includes the following steps: S1: According to the mass ratio, chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate: deionized water = (1 - 1.5): (18 - 22): (0.5 - 0.8): (80 - 90), homogeneously prepare the aqueous phase matrix; S2: According to the mass ratio, sesame oil: beeswax: poly (lactic-co-glycolic acid): dimethicone = (90 - 100): (3 - 5): (8 - 10): (0.3 - 0.6), dissolve the beeswax in the sesame oil to obtain beeswax-modified sesame oil, and then add poly (lactic-co-glycolic acid) and dimethicone, and perform ultrasonic emulsification to obtain the oil phase matrix; S3: According to the mass ratio, aqueous phase matrix: oil phase matrix: lecithin = (7 - 8): (4 - 5): (0.3 - 0.5) for compounding to prepare the biphasic adhesion matrix; S4: According to the mass parts, add barnacle peptide, lactocin, resveratrol, and vitamin B group to the biphasic adhesion matrix, mix evenly, and finally add hydroxyapatite, mix evenly, and evaporate to obtain a paste-like drug.

[0013] In S2 of the above drug preparation method, the temperature of ultrasonic emulsification is 55°C - 65°C, the power of ultrasonic emulsification is 300W - 350W, and the frequency of ultrasonic emulsification is 30kHz - 40kHz.

[0014] In S3 of the above drug preparation method, the compounding is to stir evenly at 300r / min - 500r / min.

[0015] In S4 of the above drug preparation method, the evaporation is to evaporate under negative pressure at 40°C - 50°C until the water content is 4wt% - 8wt%.

[0016] The oral mucosa repair drug and its preparation method provided by the present invention have the following beneficial effects: First, in the preparation of barnacle peptide, pepsin preliminarily enzymatically hydrolyzes the protein in the goose barnacle soft body under acidic conditions and decomposes it into specific peptide segments; then, earthworm kinase is used in a neutral environment to further enzymatically hydrolyze to obtain small peptides with specific effects. During the cell repair process, it activates the relevant signal pathways in the cells, promotes the expression of genes related to cell proliferation, accelerates the cell cycle process, and thus promotes cell proliferation; at the same time, it has antioxidant and antibacterial activities. Barnacle peptide has good biocompatibility, can promote the proliferation and repair of oral mucosa cells, and participates in various physiological mechanisms in the oral mucosa repair process.

[0017] II. In the preparation of lactobacillin, celery juice and bitter gourd juice provide additional nutrients and growth factors for Lactobacillus salivarius, promoting its growth and bacteriocin production, and endowing lactobacillin with some additional biological activities. Glucose oxidase helps maintain the redox state in the culture medium, promotes the metabolic activities of Lactobacillus salivarius, and increases the yield of lactobacillin. Lactobacillin can regulate the immune response of cells, inhibit inflammatory reactions, reduce cell damage caused by inflammation, and create a favorable environment for cell repair. Lactobacillin has antibacterial effects, can inhibit harmful microorganisms in the oral cavity, maintain the balance of oral microorganisms, reduce infections, and is beneficial to the repair of oral mucosa.

[0018] III. In the preparation of the biphasic adhesion matrix, the chondroitin sulfate-quaternary ammonium salt chitosan complex has good biocompatibility and adhesiveness, can interact with biomolecules on the surface of oral mucosa, and achieve firm adhesion of drugs to the oral mucosa. Poloxamer 407 regulates the rheological properties of the matrix, making it a liquid at room temperature for easy mixing and use, and transforming into a gel-like state at body temperature, which is beneficial to the retention of drugs on the surface of oral mucosa. Sodium alginate increases the viscosity and stability of the matrix, helps form a uniform matrix structure, and improves the adhesiveness and sustained-release performance of drugs. Beeswax is dissolved in sesame oil to form beeswax-modified sesame oil, which is the main component of the oil phase, provides a hydrophobic environment, is beneficial to the sustained release of drugs and protects drug components. Poly(lactic-co-glycolic acid) has good biodegradability and biocompatibility, regulates the physical properties of the oil-phase matrix, controls the drug release rate, and can also assist in improving the adhesiveness of the biphasic adhesion matrix. Dimethicone reduces the surface tension of the oil-phase matrix, makes it better mix with the water-phase matrix, and helps drugs distribute evenly on the surface of oral mucosa. Lecithin, as an emulsifier, promotes the emulsification of the water-phase matrix and the oil-phase matrix, forms a stable biphasic structure, and improves the stability and adhesiveness of the matrix. The biphasic adhesion matrix prepared by formulating each component in a certain proportion has good biocompatibility and adhesiveness, can form a stable drug carrier on the surface of oral mucosa, achieve slow drug release, and prolong the action time of drugs at the damaged site.

[0019] IV. In the preparation of chondroitin sulfate-chitosan quaternary ammonium salt complex, chondroitin sulfate has hydrophilicity and biological activity, and can form a complex with chitosan quaternary ammonium salt through electrostatic binding, providing good biocompatibility and biological activity for the complex. Chitosan quaternary ammonium salt has antibacterial and cationic properties, can undergo electrostatic binding with the anionic groups of chondroitin sulfate, and at the same time improve the adhesion and antibacterial properties of the complex. Polyethylene glycol increases the viscosity of the solution, is conducive to the interaction between molecules, and increases the entanglement and flexibility of molecular chains. Sodium chloride regulates the ionic strength of the solution, promotes the occurrence of electrostatic binding, and makes the complex form a more compact network structure. The chondroitin sulfate-chitosan quaternary ammonium salt complex combines the advantages of chondroitin sulfate and chitosan quaternary ammonium salt, has good biocompatibility, adhesion and antibacterial properties, and can play a variety of roles in oral mucosa repair, including promoting cell adhesion and proliferation, inhibiting bacterial growth, etc.

[0020] V. Resveratrol: It has anti-inflammatory and antioxidant effects, can reduce the inflammatory response of oral mucosa, protect oral mucosa cells from oxidative damage, and promote the repair of oral mucosa. Hydroxyapatite can enhance the mechanical properties of drugs, and at the same time help to promote the attachment and growth of oral mucosa cells and participate in the repair process of oral mucosa. Vitamin B group: Participates in the metabolic process of cells, promotes the growth and repair of oral mucosa cells, and maintains the normal physiological functions of oral mucosa. Specific embodiments

[0021] The present invention will be further described below in conjunction with specific implementation cases, but the present invention is not limited to these embodiments.

[0022] Example 1: A drug for repairing oral mucosa is prepared from the following raw materials in parts by mass: 2 parts of barnacle peptide, 1 part of lactocin, 0.5 part of resveratrol, 70 parts of biphasic adhesion matrix, 5 parts of hydroxyapatite, 0.5 part of vitamin B group, and the balance is deionized water with a water content of 4 wt%; the vitamin B group is an equimass ratio mixture of VB1, VB2, VB6 and VB12.

[0023] Among them, the preparation method of barnacle peptide includes: crushing the goose barnacle mollusk to obtain a crushed product, adding a pH 1.5 hydrochloric acid aqueous solution 8 times the mass of the crushed product, adding 1% pepsin based on the mass of the crushed product, enzymolyzing at 35°C for 1 h, adjusting the pH to 7, adding 1.5% earthworm kinase based on the mass of the crushed product, enzymolyzing at 35°C for 1.5 h, inactivating the enzyme at 85°C for 10 min, centrifuging at 4000 r / min for 10 min, taking the supernatant, and performing ultrafiltration using an ultrafiltration membrane to obtain a retentate between 1 kDa and 3 kDa, and freeze-drying to obtain barnacle peptide.

[0024] Among them, the preparation method of lactobacillin includes: adding 2 wt% activated Lactobacillus salivarius, 1 wt% celery juice, 0.5 wt% balsam pear juice and 0.1 wt% glucose oxidase into MRS medium, anaerobically culturing at 36 °C for 48 h to obtain a bacterial liquid, centrifuging at 6000 r / min for 15 min, collecting the supernatant, adding 50 wt% ammonium sulfate based on the mass of the supernatant, standing at 4 °C for 12 h, precipitating the bacteriocin, centrifuging at 8000 r / min for 20 min, collecting the precipitate, using a dialysis bag with a molecular weight cut-off of 1 kDa, dialyzing in flowing deionized water for 12 h, and freeze-drying to obtain lactobacillin.

[0025] The preparation method of the above-mentioned oral mucosa repair drug includes the following steps: S1: According to the mass ratio, chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate: deionized water = 1:18:0.5:80, homogenizing and preparing an aqueous phase matrix; S2: According to the mass ratio, sesame oil: beeswax: poly(lactic-co-glycolic acid): dimethicone = 90:3:8:0.3, dissolving beeswax in sesame oil to obtain beeswax-modified sesame oil, then adding poly(lactic-co-glycolic acid) and dimethicone, and performing ultrasonic emulsification at a temperature of 55 °C, a power of 300 W, and a frequency of 30 kHz to obtain an oil phase matrix; S3: According to the mass ratio, aqueous phase matrix: oil phase matrix: lecithin = 7:4:0.3, stirring and compounding evenly at 300 r / min to prepare a biphasic adhesive matrix; S4: According to the mass parts, adding barnacle peptide, lactobacillin, resveratrol, and vitamin B group into the biphasic adhesive matrix, mixing evenly, and finally adding hydroxyapatite, mixing evenly, and evaporating under negative pressure at 40 °C until the water content is 4 wt% to obtain a paste-like drug.

[0026] Among them, the composite mass ratio of chondroitin sulfate to quaternary ammonium chitosan in the chondroitin sulfate - quaternary ammonium chitosan complex is 1.5:0.18; the preparation method includes: by mass parts: dissolving 1.5 parts of chondroitin sulfate in 80 parts of phosphate buffer solution with pH 5.3 to form solution A; dissolving 0.18 parts of quaternary ammonium chitosan in 50 parts of phosphate buffer solution with pH 5.3, then adding 0.5 parts of polyethylene glycol and 0.3 parts of sodium chloride, stirring evenly at 200 r / min to form solution B; under stirring at 200 r / min, dropping solution B into solution A at a speed of 1 mL / min. After the dropping is completed, continue to stir and react at 200 r / min for 1 h to fully carry out electrostatic binding to obtain a reaction solution, and let it stand at low temperature of 4°C for 2 h to promote the molecular chain to shrink and entangle, and physically embed the electrostatically bound quaternary ammonium chitosan molecules in the chondroitin sulfate network structure; use a dialysis bag with a cut-off molecular weight of 1 kDa to dialyze in flowing deionized water for 12 h, and then freeze-dry to obtain the chondroitin sulfate - quaternary ammonium chitosan complex.

[0027] Example 2: A drug for repairing oral mucosa is prepared from the following raw materials by mass parts: 3 parts of barnacle peptide, 2 parts of lactocillin, 1.2 parts of resveratrol, 78 parts of a biphasic adhesion matrix, 7.5 parts of hydroxyapatite, 1.5 parts of vitamin B complex, and the balance is deionized water with a water content of 5 wt%; the vitamin B complex is a mixture of VB1, VB2, VB6, and VB12 in a mass ratio of 1:1:2:2.

[0028] Among them, the preparation method of barnacle peptide includes: crushing the gooseneck barnacle mollusk to obtain a crushed product, adding a hydrochloric acid aqueous solution with pH 2 that is 9 times the mass of the crushed product, adding pepsin that is 1.5% of the mass of the crushed product, enzymolyzing at 37°C for 1.5 h, adjusting the pH to 7.5, adding lumbrokinase that is 2% of the mass of the crushed product, enzymolyzing at 37°C for 1.5 h, inactivating the enzyme at 88°C for 12 min, centrifuging at 4500 r / min for 12 min, taking the supernatant, and performing ultrafiltration using an ultrafiltration membrane to obtain the retentate between 1 kDa and 3 kDa, and then freeze-drying to obtain barnacle peptide.

[0029] Among them, the preparation method of lactocillin includes: adding 2.5 wt% of activated Lactobacillus salivarius, 1.2 wt% of celery juice, 0.8 wt% of balsam pear juice, and 0.15 wt% of glucose oxidase to the MRS medium, anaerobically culturing at 37°C for 60 h to obtain a bacterial liquid, centrifuging at 7000 r / min for 18 min, collecting the supernatant, adding ammonium sulfate that is 55 wt% of the mass of the supernatant, standing at 5°C for 15 h, precipitating the bacteriocin, centrifuging at 9000 r / min for 25 min, collecting the precipitate, using a dialysis bag with a cut-off molecular weight of 1 kDa to dialyze in flowing deionized water for 18 h, and then freeze-drying to obtain lactocillin.

[0030] The preparation method of the above-mentioned oral mucosa repair drug comprises the following steps: S1: According to the mass ratio, chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate: deionized water = 1.2: 20: 0.7: 85, homogeneously prepare the aqueous phase matrix; S2: According to the mass ratio, sesame oil: beeswax: poly (lactic-co-glycolic acid): dimethicone = 95: 4: 9: 0.5, dissolve the beeswax in the sesame oil to obtain beeswax-modified sesame oil, then add poly (lactic-co-glycolic acid) and dimethicone, and perform ultrasonic emulsification at a temperature of 60 °C, a power of 350 W, and a frequency of 30 kHz to obtain the oil phase matrix; S3: According to the mass ratio, aqueous phase matrix: oil phase matrix: lecithin = 7.5: 4.5: 0.4, stir and compound evenly at 400 r / min to prepare the biphasic adhesion matrix; S4: According to the mass parts, add barnacle peptide, lactobacillin, resveratrol, and vitamin B group to the biphasic adhesion matrix, mix evenly, finally add hydroxyapatite, mix evenly, and evaporate under negative pressure at 45 °C until the water content is 5 wt% to obtain a paste-like drug.

[0031] Among them, the composite mass ratio of chondroitin sulfate and chitosan quaternary ammonium salt in the chondroitin sulfate-chitosan quaternary ammonium salt complex is 1.7: 0.21; the preparation method includes: by mass parts: dissolve 1.7 parts of chondroitin sulfate in 90 parts of phosphate buffer solution with pH 5.5 to form solution A; dissolve 0.21 parts of chitosan quaternary ammonium salt in 55 parts of phosphate buffer solution with pH 5.5, then add 0.8 parts of polyethylene glycol and 0.4 parts of sodium chloride, and stir evenly at 250 r / min to form solution B; under stirring at 250 r / min, add solution B dropwise to solution A at a speed of 1.5 mL / min. After the dropwise addition is completed, continue to stir and react at 250 r / min for 1 h to fully carry out electrostatic binding to obtain the reaction solution, and stand still at 5 °C for 2.5 h to promote the contraction and entanglement of the molecular chains, and physically embed the electrostatically bound chitosan quaternary ammonium salt molecules in the chondroitin sulfate network structure; use a dialysis bag with a cut-off molecular weight of 1 kDa, dialyze in flowing deionized water for 18 h, and freeze-dry to obtain the chondroitin sulfate-chitosan quaternary ammonium salt complex.

[0032] Example 3: An oral mucosa repair drug is made from the following raw materials in mass parts: 5 parts of barnacle peptide, 3 parts of lactobacillin, 2 parts of resveratrol, 85 parts of biphasic adhesion matrix, 10 parts of hydroxyapatite, 3 parts of vitamin B group, and the balance is deionized water with a water content of 8 wt%; the vitamin B group is a mixture of VB1, VB2, VB6, and VB12 in a mass ratio of 1: 3: 1: 2.

[0033] Among them, the preparation method of barnacle peptide includes: crushing the barnacle soft body to obtain a crushed product, adding a hydrochloric acid aqueous solution with a pH of 2.5 that is 10 times the mass of the crushed product, adding pepsin that is 2% of the mass of the crushed product, enzymolyzing at 40 °C for 2 h, adjusting the pH to 8, adding lumbrokinase that is 3% of the mass of the crushed product, enzymolyzing at 40 °C for 2 h, inactivating the enzyme at 90 °C for 15 min, centrifuging at 5000 r / min for 15 min, taking the supernatant, performing ultrafiltration using an ultrafiltration membrane to obtain a retentate between 1 kDa and 3 kDa, and freeze-drying to obtain barnacle peptide.

[0034] Among them, the preparation method of lactobacillin includes: adding 3 wt% activated Lactobacillus salivarius, 1.5 wt% celery juice, 1 wt% bitter gourd juice, and 0.2 wt% glucose oxidase to MRS medium, anaerobically culturing at 38 °C for 72 h to obtain a bacterial solution, centrifuging at 8000 r / min for 20 min, collecting the supernatant, adding ammonium sulfate that is 60 wt% of the mass of the supernatant, standing at 6 °C for 18 h, precipitating the bacteriocin, centrifuging at 10000 r / min for 30 min, collecting the precipitate, using a dialysis bag with a molecular weight cut-off of 1 kDa, dialyzing in flowing deionized water for 24 h, and freeze-drying to obtain lactobacillin.

[0035] The preparation method of the above-mentioned oral mucosa repair drug includes the following steps: S1: According to the mass ratio, chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate: deionized water = 1.5: 22: 0.8: 90, homogenize and prepare an aqueous phase matrix; S2: According to the mass ratio, sesame oil: beeswax: poly (lactic-co-glycolic acid): dimethicone = 100: 5: 10: 0.6, dissolve the beeswax in the sesame oil to obtain beeswax-modified sesame oil, then add poly (lactic-co-glycolic acid) and dimethicone, and perform ultrasonic emulsification at a temperature of 65 °C, a power of 350 W, and a frequency of 40 kHz to obtain an oil phase matrix; S3: According to the mass ratio, aqueous phase matrix: oil phase matrix: lecithin = 8: 5: 0.5, stir and compound evenly at 500 r / min to prepare a biphasic adhesive matrix; S4: According to the mass parts, add barnacle peptide, lactobacillin, resveratrol, and vitamin B group to the biphasic adhesive matrix, mix evenly, finally add hydroxyapatite, mix evenly, and evaporate under negative pressure at 50 °C until the water content is 8 wt% to obtain a paste-like drug.

[0036] The composite mass ratio of chondroitin sulfate to chitosan quaternary ammonium salt in the chondroitin sulfate-chitosan quaternary ammonium salt complex is 2.0:0.25; the preparation method comprises: dissolving 2.0 parts of chondroitin sulfate in 100 parts of pH 5.6 phosphate buffer to form solution A; dissolving 0.25 parts of chitosan quaternary ammonium salt in 60 parts of pH 5.6 phosphate buffer, then adding 1.0 parts of polyethylene glycol and 0.5 parts of sodium chloride, stirring at 250r / min to form solution B; Under stirring at 0 r / min, solution B was added dropwise to solution A at a speed of 2 mL / min. After the addition was completed, the stirring was continued at 250 r / min for 1.5 h to allow sufficient electrostatic binding to occur, thereby obtaining a reaction solution. The reaction solution was allowed to stand at a low temperature of 6°C for 3 h to promote molecular chain contraction and entanglement, thereby physically embedding the electrostatically bound chitosan quaternary ammonium salt molecules in the chondroitin sulfate network structure. A dialysis bag with a molecular weight cutoff of 1 kDa was used for dialysis in flowing deionized water for 24 h, and the solution was freeze-dried to obtain a chondroitin sulfate-chitosan quaternary ammonium salt complex.

[0037] Sources of raw materials in the above examples: The enzymatic activity of pepsin is 1000 NFu / mg, from Shaanxi Guanchen Biotechnology Co., Ltd. The enzymatic activity of lumbrokinase is 20,000 IU / mg, from Lanli Biotechnology (Xi'an) Co., Ltd. The bacterial activity of Lactobacillus salivarius is 10 billion CFU / g, from Xi'an Miaoguo Biotechnology Co., Ltd. The enzymatic activity of glucose oxidase is U / g, from Zhejiang Fuxuan Biotechnology Co., Ltd. The purity of resveratrol is 98%, from Shanxi Qixin Biotechnology Co., Ltd. The particle size of hydroxyapatite is less than 80 μm, from Lanli Biotechnology (Xi'an) Co., Ltd. Chondroitin sulfate is derived from shark bone, from Shaanxi Taike Biotechnology Co., Ltd. Chitosan quaternary ammonium salt is hydroxypropyltrimethylammonium chloride chitosan, with a purity of 99%, from Xi'an Hols Peptide Bioengineering Co., Ltd. Polyethylene glycol is pharmaceutical grade polyethylene glycol 6000, from Guangzhou Cancheng Chemical Technology Co., Ltd. Poloxamer 407 is pharmaceutical grade imported from BASF, from Shaanxi Zhengyi Pharmaceutical Excipients Co., Ltd. Sodium alginate is food grade, from Fujian Rongsen Biotechnology Co., Ltd. Beeswax is pharmaceutical grade, melting point 60°C, from Shanghai Gaoming Chemical Co., Ltd. Poly(lactic-co-glycolic acid) copolymer is pharmaceutical grade, model PLG50-04, from Wuhan Kemik Biopharmaceutical Technology Co., Ltd. Dimethicone is pharmaceutical grade, model 750cs, from Shaanxi Zhengyi Pharmaceutical Excipients Co., Ltd. Lecithin is soybean lecithin, from Nanjing Yishengyuan Biotechnology Co., Ltd.

[0038] Comparative Example 1 In the preparation of barnacle peptides, lumbrokinase was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0039] Comparative Example 2 In the preparation of barnacle peptides, lumbrokinase was replaced by papain (enzyme activity 100,000 U / g); other parameters and methods were the same as in Example 1.

[0040] Comparative Example 3 In the preparation of barnacle peptide, pepsin was not used for enzymatic hydrolysis; other parameters and methods were the same as in Example 1.

[0041] Comparative Example 4 In the preparation of barnacle peptide, pepsin was replaced with trypsin (enzyme activity 4000 U / g), and the 1.5 pH hydrochloric acid aqueous solution was replaced with an 8.0 pH sodium hydroxide aqueous solution; other parameters and methods were the same as in Example 1.

[0042] Comparative Example 5 In the preparation of lactobacillin, 1 wt% celery juice and 0.5 wt% bitter gourd juice were not added; other parameters and methods were the same as in Example 1.

[0043] Comparative Example 6 The chondroitin sulfate-chitosan quaternary ammonium salt complex was directly replaced with chondroitin sulfate and chitosan quaternary ammonium salt (without electrostatic and embedding binding); other parameters and methods were the same as in Example 1.

[0044] Comparative Example 7 In the preparation of the chondroitin sulfate-chitosan quaternary ammonium salt complex, polyethylene glycol was not added; other parameters and methods were the same as in Example 1.

[0045] Comparative Example 8 In the preparation of the chondroitin sulfate-chitosan quaternary ammonium salt complex, polyethylene glycol and sodium chloride were not added; other parameters and methods were the same as in Example 1.

[0046] Comparative Example 9 In the preparation of the chondroitin sulfate-chitosan quaternary ammonium salt complex, low-temperature static setting was not carried out; other parameters and methods were the same as in Example 1.

[0047] Comparative Example 10 In the aqueous phase matrix, the chondroitin sulfate-chitosan quaternary ammonium salt complex was not added; other parameters and methods were the same as in Example 1.

[0048] Comparative Example 11 In the oil phase matrix, poly(lactic-co-glycolic acid) was not added; other parameters and methods were the same as in Example 1.

[0049] I. Adhesion time detection Artificial simulated saliva preparation: 10.5 g of NaCl, 2.5 g of Na2HPO4, and 0.2 g of KH2PO4 were dissolved in 1000 mL of water. Take porcine large intestine mucosa with a diameter of 1.5 cm to simulate oral mucosa. After soaking in artificial simulated saliva for 2 h, it was fixed on a glass slide, keeping the mucosal surface flat. Take the repair drugs (the repair drugs of Examples 1 to 3 and Comparative Examples 6 to 11 respectively) and evenly coat them on the mucosa to form a coating with a thickness of 1 mm. Let it stand for 3 min, and then soak it in a beaker containing 50 mL of artificial simulated saliva. The beaker was placed in a 37 °C water bath, and the time for the repair drug to completely fall off from the porcine large intestine mucosa, that is, the adhesion time, was observed and recorded every 5 min.

[0050] Table 1 Detection results of adhesion time (average value of 3 parallel samples)

[0051] From the above results, it can be seen that the repair drugs of Examples 1 to 3 have good adhesion. Among them, the chondroitin sulfate-chitosan quaternary ammonium salt complex forms a stable structure through electrostatic binding and physical embedding. Chondroitin sulfate carries a negative charge, and chitosan quaternary ammonium salt carries a positive charge. In a phosphate buffer solution with a specific pH, they bind to each other through electrostatic attraction; the addition of polyethylene glycol and sodium chloride, polyethylene glycol increases the entanglement and flexibility of the molecular chain, and sodium chloride adjusts the ionic strength to promote the interaction between molecules, making the complex form a more compact network structure. Poloxamer 407 reduces the interfacial tension, enabling the matrix to spread better on the mucosal surface; sodium alginate has good film-forming property and viscosity, and synergizes with the chondroitin sulfate-chitosan quaternary ammonium salt complex to form a continuous and firm adhesion layer on the mucosal surface, enhancing the intermolecular force and prolonging the adhesion time.

[0052] In Comparative Example 6, electrostatic binding and physical embedding of chondroitin sulfate and chitosan quaternary ammonium salt were not carried out, and the two components existed in a simple mixed form. In the simulated saliva environment, due to the lack of stable binding force, they were easily dispersed under the action of water molecules and could not form an effective adhesion network structure. Compared with the examples where complexes were formed, the binding force with the mucosal surface was greatly weakened, and the drug would be washed off by simulated saliva in a short time, resulting in a significantly shortened adhesion time.

[0053] In Comparative Example 7, polyethylene glycol plays a role in solubilization, thickening, and promoting intermolecular entanglement in the preparation of the complex. After the absence of polyethylene glycol, the entanglement degree between chondroitin sulfate and chitosan quaternary ammonium salt molecules decreases, the structure of the complex becomes loose, and the stability decreases. This unstable complex in simulated saliva is easily disrupted by the intermolecular interaction, resulting in an accelerated rate of drug detachment from the mucosal surface. Therefore, the adhesion time is shorter than that of the examples, but due to the still existing electrostatic binding, its adhesion performance is better than that of Comparative Example 6 with simple mixing.

[0054] In Comparative Example 8, both polyethylene glycol and sodium chloride were absent. Sodium chloride affects the intermolecular electrostatic interaction and promotes the formation of the complex by adjusting the ionic strength during the preparation of the complex. The simultaneous absence of both seriously disrupted the formation process of the complex. On the one hand, the entanglement and binding force between molecules were weakened, and on the other hand, the electrostatic interaction could not be effectively regulated and stabilized. As a result, the structure of the complex was extremely unstable, easily disintegrated in simulated saliva, the adhesion performance of the drug decreased, and it was shorter than that of Comparative Example 7 where only polyethylene glycol was absent.

[0055] In Comparative Example 9, the low-temperature standing step is crucial for the formation of the complex. Under low-temperature conditions, the molecular motion slows down, which is beneficial for the contraction and entanglement of molecular chains, enabling the electrostatically bound chitosan quaternary ammonium salt molecules to be better physically embedded in the chondroitin sulfate network structure to form a more compact and stable complex. Without low-temperature standing, the structure of the complex is not tight enough, the binding between molecules is not firm enough, and under the soaking and scouring of simulated saliva, the complex is prone to looseness, and the time for the drug to fall off from the mucosal surface is advanced, resulting in a shortened adhesion time. However, due to the still existing electrostatic binding and partial molecular entanglement, its adhesion time is longer than that of Comparative Examples 6 to 8.

[0056] In Comparative Example 10, the chondroitin sulfate-chitosan quaternary ammonium salt complex in the aqueous matrix is the adhesion component of the biphasic adhesion matrix. After the absence of this complex, the adhesion performance of the aqueous matrix is basically lost, and an effective adhesion layer cannot be formed only by poloxamer 407 and sodium alginate. Poloxamer 407 mainly plays an emulsifying and interfacial tension reducing role. Although sodium alginate has a certain viscosity, without the synergism of the complex, it cannot form a firm adhesion structure on the mucosal surface. The drug can hardly adhere in simulated saliva, so the adhesion time is shortened.

[0057] In Comparative Example 11, poly(lactic-co-glycolic acid) is an important component of the oil-phase matrix, which has good film-forming and adhesion properties, and can enhance the stability of the oil-phase matrix and the binding force with the aqueous matrix. Without adding poly(lactic-co-glycolic acid), the structure and properties of the oil-phase matrix change, its stability decreases, and the binding with the aqueous matrix is not tight enough, resulting in the overall performance of the biphasic adhesion matrix being affected. Although other components can still provide a certain adhesion force, it cannot reach the adhesion effect of the biphasic matrix in the examples, and the adhesion time of the drug on the mucosal surface is shortened.

[0058] II. Detection of in vitro mucosal cell repair ability For precise testing, the samples of the repair drug only contain the active ingredients of barnacle peptide, salivaricin, and resveratrol. Samples of the active ingredients of the repair drugs of Examples 1 to 3 and Comparative Examples 1 to 5 were prepared respectively. Human immortalized oral keratinocytes (HOK cells) were taken and placed in DMEM medium containing 10% fetal bovine serum and 1% double antibiotics (penicillin-streptomycin), and cultured in an incubator at 37 °C and 5% CO2 until the logarithmic growth phase. Well-grown HOK cells were digested with 0.25% trypsin, and then the cell density was adjusted to 1×10 5 cells / mL, and inoculated into a 24-well plate, 1 mL per well. After culturing for 24 h until the cells adhered to the wall, the medium was discarded, and the cells were washed twice with PBS, and then a medium containing 1 mM H2O2 was added and treated for 2 h to establish an oxidative damage model. The medium containing H2O2 in the 24-well plate was discarded, and the cells were washed three times with PBS, and then a medium containing 100 μg / mL of the repair drug sample was added, 1 mL per well, and three replicate wells were set in each group. After the drug treatment for 24 h, 20 μL of CCK-8 solution was added to each well, and the incubation was continued for 4 h, and the absorbance (OD value) was measured at 450 nm with an enzyme-labeled instrument. In addition, the cell culture supernatant was collected, centrifuged at 1200 rpm for 10 min to obtain the centrifugate, and the contents of inflammatory factors IL-6 and IL-8 were detected using an ELISA kit.

[0059] Table 2 Detection results of in vitro mucosal cell repair ability (mean value) The above results demonstrate that the repair drugs of Examples 1 to 3 possess excellent cell damage repair capabilities and promote anti-inflammatory effects. Barnacle peptides contain multiple active peptide segments. During the cell repair process, they activate relevant signaling pathways within cells, promote the expression of cell proliferation-related genes, accelerate cell cycle progression, and thus promote cell proliferation. Furthermore, they possess antioxidant activity, scavenging excess reactive oxygen species (ROS) produced by H₂O₂ in cells, reducing ROS damage to cellular DNA, proteins, and lipids, protecting cell structure and function, and improving cell survival. Salivaricin modulates cellular immune responses, inhibits inflammatory reactions, reduces inflammatory damage to cells, and creates a favorable environment for cell repair. Resveratrol activates the Nrf2 signaling pathway, upregulating the expression of antioxidant enzymes and enhancing cellular antioxidant capacity. It also inhibits the NF-κB signaling pathway and reduces the production of inflammatory factors. In the H₂O₂-induced oxidative damage model, elevated intracellular oxidative stress levels activate inflammatory signaling pathways such as NF-κB, leading to the massive synthesis and secretion of inflammatory factors such as IL-6 and IL-8. The combination of barnacle peptide, salivaricin, and resveratrol in the examples can inhibit activation of the NF-κB signaling pathway and reduce the transcription and expression of inflammatory factor genes. Barnacle peptide blocks upstream signaling by binding to related receptors; salivaricin regulates immune cell function and reduces the release of inflammatory mediators; and resveratrol inhibits the activity of IκB kinase (IKK), preventing the phosphorylation and activation of NF-κB, preventing it from entering the cell nucleus and initiating transcription of inflammatory factor genes.

[0060] In comparative example 1, lumbrokinase is not used for enzymatic hydrolysis in the preparation of barnacle peptides. Pepsin can only perform preliminary decomposition of the gooseneck barnacle soft body and cannot fully break specific peptide bonds, resulting in the inability to completely release peptide segments with repair activity. The generated barnacle peptides have low activity and cannot effectively activate the repair signal pathway in the cell. After the cells are damaged by H2O2 oxidation, their proliferation and repair capabilities are insufficient, a large number of cells die, and the number of surviving cells decreases, so the OD value decreases. At the same time, due to the lack of effective repair and anti-inflammatory active ingredients, the inflammatory signal pathway in the cell continues to be activated, NF-κB enters the cell nucleus in large quantities, and initiates the transcription of inflammatory factor genes such as IL-6 and IL-8, resulting in the secretion of large amounts of inflammatory factors, and their content is significantly increased.

[0061] Comparative Example 2: The enzymatic cleavage site and mechanism of action of papain are different from those of lumbrokinase. Its enzymatic hydrolysis of gooseneck barnacles is not as expected, and the structure and activity of the resulting barnacle peptides are changed. These abnormal barnacle peptides cannot effectively bind to the corresponding receptors or signaling molecules in the cells and cannot exert efficient pro-repair and anti-inflammatory functions. Under oxidative damage, the cells suffer more damage, the repair process is hindered, and the cell survival rate is reduced. The inflammatory response is not controlled, the cells remain in an inflammatory state, and the secretion of inflammatory factors such as IL-6 and IL-8 increases.

[0062] In Comparative Example 3, pepsin was not used for enzymatic hydrolysis, and only the subsequent lumbrokinase could not effectively decompose the goose barnacle raw material. Pepsin preliminarily hydrolyzes the proteins in the raw material in an acidic environment, providing a suitable substrate structure for the further action of lumbrokinase. Without the action of pepsin, lumbrokinase is difficult to play its role, and almost no active barnacle peptides can be generated. After the cells were damaged by H2O2 oxidation, they completely lost the repair support of barnacle peptides, and the cell damage was severe. The inflammatory response was intense, the intracellular inflammatory signaling pathway was overactivated, and a large amount of IL-6 and IL-8 were released, and their contents reached a relatively high level.

[0063] In Comparative Example 4, pepsin was replaced with trypsin, and the reaction pH value changed from acidic to alkaline. After changing the reaction environment, it was impossible to effectively decompose the goose barnacle raw material like pepsin. At the same time, different enzyme digestion methods led to the generated barnacle peptides being not only in small amounts, but also severely damaged in structure and activity, and unable to exert normal repair and anti-inflammatory functions. In this case, the repair ability of the cells was almost lost, and under H2O2 oxidative damage, the survival rate was extremely low, and the OD value was the lowest. The inflammatory response was completely out of control, and a large amount of inflammatory factors such as IL-6 and IL-8 were synthesized and secreted, and their contents reached the highest level.

[0064] Celery juice and bitter gourd juice in Comparative Example 5 contain special components that promote the growth and metabolism of Lactobacillus salivarius. These components can regulate the metabolic pathway of Lactobacillus salivarius and promote the synthesis and secretion of salivaricin. Without adding celery juice and bitter gourd juice, the growth of Lactobacillus salivarius was affected to a certain extent, and the yield, purity, and activity of salivaricin decreased, and its antibacterial and anti-inflammatory effects weakened. Although barnacle peptides and resveratrol can still play a certain role, the repair and anti-inflammatory effects of the overall drug decreased. The repair ability of the cells after oxidative damage was not as good as that in the examples, and the number of surviving cells decreased, and the OD value was lower. The secretion of inflammatory factors increased slightly, and the contents of IL-6 and IL-8 were higher than those in the examples but lower than those in Comparative Examples 1 to 4 because there were other active components that partially maintained the anti-inflammatory function.

[0065] III. Detection of antibacterial effect After activating Staphylococcus aureus (DSM45902), Helicobacter pylori (ATCC43504), and Candida albicans (ATCC10231), the concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL. Take 0.1 mL of the bacterial suspension and evenly coat it on the surface of the corresponding solid medium. Use a punch to punch holes with a diameter of 6 mm on the medium, and suck 20 μL of the repair drug solution (the repair drugs of the whole components of Examples 1 to 3 and Comparative Examples 1 to 5 were respectively diluted with physiological saline to a concentration of 500 mg / mL) and add it to the holes. After culturing for 48 h in an adapted environment, measure the diameter of the antibacterial zone. There are three parallel samples in each group.

[0066] Table 3 Antibacterial effect test results (average value) From the above results, it can be seen that the repair drugs of Examples 1 to 3 have good antibacterial effects.

[0067] Comparative Examples 1 to 5: Mainly affect the preparation process of barnacle peptide and salivaricin, resulting in changes in active ingredients or reduced content. In Comparative Example 1, the activity of barnacle peptide was reduced due to the lack of lumbrokinase enzymatic hydrolysis; in Comparative Example 2, papain replaced lumbrokinase, resulting in abnormal structure of barnacle peptide; in Comparative Example 3, the lack of pepsin resulted in the ineffective production of barnacle peptide; in Comparative Example 4, trypsin and pH changes almost lost the activity of barnacle peptide; in Comparative Example 5, the lack of celery juice and bitter melon juice resulted in decreased production, purity, and activity of salivaricin. These changes in active ingredients weakened the inhibitory effect of the drug on pathogens. In the absence of effective inhibition, the growth of Staphylococcus aureus, Helicobacter pylori, and Candida albicans was less restricted, and the diameter of the inhibition zone was significantly smaller than that of the embodiment.

Claims

1. A repair drug for oral mucosa, characterized in that, The drug is prepared from the following raw materials in parts by mass: 2 to 5 parts of barnacle peptide, 1 to 3 parts of lactobacillin, 0.5 to 2 parts of resveratrol, 70 to 85 parts of a biphasic adhesion matrix, 5 to 10 parts of hydroxyapatite, 0.5 to 3 parts of vitamin B group, and the balance is deionized water with a water content of 4wt% to 8wt%; The barnacle peptide contains the product between 1 kDa and 3 kDa obtained by enzymatically hydrolyzing the crushed soft body of goose barnacle successively with pepsin and earthworm kinase; The component mass ratio of the biphasic adhesion matrix is aqueous phase matrix: oil phase matrix: lecithin = (7 - 8):(4 - 5):(0.3 - 0.5); the component mass ratio of the aqueous phase matrix is chondroitin sulfate-chitosan quaternary ammonium salt complex: poloxamer 407: sodium alginate = (1 - 1.5):(18 - 22):(0.5 - 0.8); the component mass ratio of the oil phase matrix is sesame oil: beeswax: poly(lactic-co-glycolic acid): dimethicone = (90 - 100):(3 - 5):(8 - 10):(0.3 - 0.6); The composite mass ratio of chondroitin sulfate and chitosan quaternary ammonium salt in the chondroitin sulfate-chitosan quaternary ammonium salt complex is (1.5 - 2.0):(0.18 - 0.25).

2. The repair drug for oral mucosa according to claim 1, wherein, The preparation method of the barnacle peptide includes: crushing the soft body of goose barnacle to obtain a crushed product, adding an aqueous hydrochloric acid solution, adding pepsin accounting for 1% - 2% of the mass of the crushed product, enzymatically hydrolyzing at 35°C - 40°C for 1h - 2h, adjusting the pH to 7 - 8, adding earthworm kinase accounting for 1.5% - 3% of the mass of the crushed product, enzymatically hydrolyzing at 35°C - 40°C for 1.5h - 2h, inactivating the enzyme, centrifuging to take the supernatant, performing ultrafiltration using an ultrafiltration membrane to obtain the retentate between 1 kDa and 3 kDa, and freeze-drying to obtain the barnacle peptide.

3. The repair drug for oral mucosa according to claim 2, characterized in that, The dosage of the aqueous hydrochloric acid solution is 8 to 10 times the mass of the crushed product; the pH value of the aqueous hydrochloric acid solution is 1.5 - 2.5; the enzyme inactivation is carried out at 85°C - 90°C for 10min - 15min; the centrifugation is carried out at 4000r / min - 5000r / min for 10min - 15min.

4. A repair drug for oral mucosa according to claim 1, characterized in that, The preparation method of the lactobacillin includes: adding 2wt% - 3wt% of activated Lactobacillus salivarius, 1wt% - 1.5wt% of celery juice, 0.5wt% - 1wt% of balsam pear juice, and 0.1wt% - 0.2wt% of glucose oxidase to the MRS medium, anaerobically culturing at 36°C - 38°C for 48h - 72h to obtain a bacterial liquid, centrifuging at 6000r / min - 8000r / min for 15min - 20min, collecting the supernatant, adding ammonium sulfate accounting for 50wt% - 60wt% of the mass of the supernatant, standing at 4°C - 6°C for 12h - 18h, precipitating the bacteriocin, centrifuging at 8000r / min - 10000r / min for 20min - 30min, collecting the precipitate, using a dialysis bag with a cut-off molecular weight of 1 kDa, dialyzing in flowing deionized water for 12h - 24h, and freeze-drying to obtain the lactobacillin.

5. A repair drug for oral mucosa according to claim 1, characterized in that The preparation method of the chondroitin sulfate-chitosan quaternary ammonium salt complex includes: By mass fraction: 1.5 to 2.0 parts of chondroitin sulfate are dissolved in 80 to 100 parts of phosphate buffer solution with a pH of 5.3 to 5.6 to form solution A; 0.18 to 0.25 parts of quaternary ammonium chitosan are dissolved in 50 to 60 parts of phosphate buffer solution with a pH of 5.3 to 5.6, then 0.5 to 1.0 parts of polyethylene glycol and 0.3 to 0.5 parts of sodium chloride are added, and stirred evenly to form solution B; under stirring, solution B is added dropwise to solution A. After the addition is completed, continue to stir and react to fully carry out electrostatic binding to obtain a reaction solution, and let it stand at low temperature to promote the shrinkage and entanglement of molecular chains, and physically embed the electrostatically bound quaternary ammonium chitosan molecules in the chondroitin sulfate network structure; use a dialysis bag with a molecular weight cut-off of 1 kDa, dialyze in flowing deionized water for 12 h to 24 h, and freeze-dry to obtain the chondroitin sulfate-quaternary ammonium chitosan complex.

6. The repair drug for oral mucosa according to claim 5, characterized in that, The stirring speed is 200 r / min to 250 r / min; the dropping speed is 1 mL / min to 2 mL / min; the reaction time is 1 h to 1.5 h; the low-temperature standing is to stand at 4 °C to 6 °C for 2 h to 3 h.

7. The repair drug for oral mucosa according to claim 1, characterized in that, The vitamin B group is a mixture of VB1, VB2, VB6 and VB12.

8. A preparation method of a repair drug for oral mucosa according to claim 1, characterized in that, It includes the following steps: S1: According to the mass ratio, chondroitin sulfate-quaternary ammonium chitosan complex: poloxamer 407: sodium alginate: deionized water = (1 - 1.5): (18 - 22): (0.5 - 0.8): (80 - 90), and homogenize to prepare an aqueous phase matrix; S2: According to the mass ratio, sesame oil: beeswax: poly (lactic-co-glycolic acid): dimethicone = (90 - 100): (3 - 5): (8 - 10): (0.3 - 0.6), dissolve beeswax in sesame oil to obtain beeswax-modified sesame oil, then add poly (lactic-co-glycolic acid) and dimethicone, and perform ultrasonic emulsification to obtain an oil phase matrix; S3: Compound according to the mass ratio of aqueous phase matrix: oil phase matrix: lecithin = (7 - 8): (4 - 5): (0.3 - 0.5) to prepare a biphasic adhesive matrix; S4: According to the mass fraction, add barnacle peptide, nisin, resveratrol, vitamin B group to the biphasic adhesive matrix, mix evenly, and finally add hydroxyapatite, mix evenly, and evaporate to obtain a paste-like drug.

9. The preparation method of a repair drug for oral mucosa according to claim 8, characterized in that, In S2, the temperature of the ultrasonic emulsification is 55 °C to 65 °C, the power of the ultrasonic emulsification is 300 W to 350 W, and the frequency of the ultrasonic emulsification is 30 kHz to 40 kHz.

10. The preparation method of a repair drug for oral mucosa according to claim 8, characterized in that, In S3, the compounding is to stir evenly at 300 r / min to 500 r / min; in S4, the evaporation is to evaporate under negative pressure at 40 °C to 50 °C until the water content is 4 wt% to 8 wt%.

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