An active pharmaceutical composition for treating oral ulcers and a preparation method thereof

Abstract

The present application relates to a kind of active pharmaceutical compositions for treating oral ulcer and its preparation method, belong to biological medicine technical field, the present application with lactoferrin peptide, spilanthes acmella willd flavone and Brazil Biji extract as main active substance, with trimethyl chitosan-fucoidan complex, high adhesion biomimetic carrier and poloxamer 407 as main adhesive and gel matrix, cooperate oligoisomaltose, menthol-β-cyclodextrin inclusion compound, ascorbic acid palmitate and glycerol etc. Preparation medicine, through the synergistic system of complex treatment, long-acting adhesion, safe control, microecological prevention, solve the core pain point that traditional oral ulcer drug action is short, side effect is big, easy recurrence. High adhesion biomimetic carrier is the four-element composite matrix of silk fibroin, hyaluronic acid-mercapto derivative, chitosan thiol modifier and pectin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an active pharmaceutical composition for treating oral ulcer and a preparation method thereof. BACKGROUND

[0002] Oral ulcer is the most common oral mucosa disease in clinic, which can occur in people of all ages. Its cause has not been completely clear. Modern medicine believes that it is related to immune function disorder, genetic factors, mental stress, nutritional deficiency, oral mucosa trauma and microbial infection, etc. The core pathological mechanism is abnormal apoptosis of oral mucosa epithelial cells, which leads to the appearance of round or oval superficial ulcer in the mucosa, accompanied by obvious burning pain.

[0003] The drug development for treating oral ulcer is carried out around three core goals of relieving symptoms, promoting healing and reducing recurrence. The drugs currently used in clinic have formed a system with multiple dosage forms and multiple action mechanisms. From the dosage form, there are two types of local medication and systemic medication. Local medication is the first choice in clinic because of its direct action on the lesion and small side effects. Common dosage forms include gargling solution, patch, gel, powder, etc. Systemic medication is mainly aimed at patients with repeated episodes and severe conditions, and mainly uses immune modulators and vitamin supplements to adjust the immune state of the body or supplement nutrition to reduce the frequency of ulcer recurrence.

[0004] From the action mechanism, the existing drugs can be divided into four categories: 1. Anti-bacterial and anti-inflammatory drugs, which can inhibit the reproduction of oral pathogenic bacteria, reduce secondary infection of ulcer and relieve inflammatory reaction; 2. Analgesics, which can block pain signal transmission through local anesthetic components to quickly reduce the burning pain; 3. Promoting healing drugs, which can supplement growth factors or provide a mucosa protective barrier to accelerate the repair of mucosa epithelial cells; 4. Immune modulators, which can regulate T lymphocyte function, improve immune disorder and reduce recurrence.

[0005] The current drugs for treating oral ulcer still have the following problems:

[0006] 1. Short action time of local medication, which needs to be used frequently: Most local drugs are easily diluted and flushed by saliva in the oral cavity, and the action time only lasts for 1-2 hours. Patients need to use them 4-6 times a day to maintain the efficacy, and the compliance is poor. Especially at night, the drug action is interrupted, which delays the healing process.

[0007] 2. Poor prevention effect of recurrence, and lack of radical treatment: There is no drug that can completely cure oral ulcer. Although systemic immune modulators can reduce the frequency of recurrence, they are only effective for 30%-40% of patients and have side effects, which cannot be widely used. Most local drugs only focus on acute treatment and have almost no preventive effect on recurrence.

[0008] 3. Some drugs have side effects, and the suitable population is limited: long-term use of chlorhexidine in mouthwash may cause tooth staining and taste changes; long-term use of hormone patches may cause oral mucosa atrophy; systemic immunomodulators have the risk of liver and kidney function damage, and are not suitable for children, pregnant women and patients with liver and kidney dysfunction.

[0009] Therefore, there is a need to develop safer, long-acting, efficient and highly adherent drugs, and to reduce recurrent infections to provide better treatment options for patients. SUMMARY

[0010] The existing technology still has the problems of short local drug action time, affecting drug efficacy, obvious side effects, and poor recurrence prevention effect. The present application provides an active pharmaceutical composition for treating oral ulcers and a preparation method thereof. The main active substances are lactoferrin peptide, spilanthes acuta flavone and brazilian bigho extract. The main adhesion and gel matrix are trimethyl chitosan-fucus polysaccharide complex, high adhesion biomimetic carrier and poloxamer 407. The drug is prepared with oligoisomaltose, menthol-β-cyclodextrin inclusion compound, ascorbic acid palmitate and glycerol. Through the synergistic system of "combined treatment + long-acting adhesion + safe regulation + microecological prevention", the core pain points of traditional oral ulcer drugs "short-acting, large side effects and easy recurrence" are solved. The specific technical scheme is as follows:

[0011] An active pharmaceutical composition for treating oral ulcers is prepared from the following raw materials in the following mass percentages: 1.0% to 3.0% lactoferrin peptide, 1.0% to 2.0% spilanthes acuta flavone, 1.0% to 1.5% brazilian bigho extract, 5.0% to 8.0% trimethyl chitosan-fucus polysaccharide complex, 8.0% to 12.0% high adhesion biomimetic carrier, 18.0% to 20.0% poloxamer 407, 3.0% to 7.0% oligoisomaltose, 0.3% to 1.0% menthol-β-cyclodextrin inclusion compound, 0.1% to 0.5% ascorbic acid palmitate, 2.0% to 5.0% glycerol, and the balance is a phosphate buffer solution with a pH of 6.8 to 7.2. The sum of the mass percentages of the above components is 100%.

[0012] The trimethyl chitosan-fucus polysaccharide complex is prepared by stirring and mixing 1.0 mg / mL to 1.5 mg / mL trimethyl chitosan solution and 1.0 mg / mL to 1.5 mg / mL fucus polysaccharide solution at a volume ratio of 1: (0.4 to 0.6), then ultrasonicating in an ice water bath, adding a sodium tripolyphosphate aqueous solution, stirring, centrifuging to obtain the precipitate, washing, and freeze-drying.

[0013] The high-adhesion biomimetic carrier is a four-component composite matrix composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier and pectin; the high-adhesion biomimetic carrier is a mixture of 4.0wt%-5.0wt% concentration of silk fibroin, 2.0wt%-3.0wt% concentration of hyaluronic acid-thiol derivative solution, 2.0wt%-2.5wt% concentration of chitosan thiol modifier solution and 2.0wt%-2.5wt% concentration of pectin solution in a volume ratio of (3-4):(2-2.5):(2-2.5):(3-4), prepared by stirring for 2-3 hours at pH 6.0-6.5;

[0014] The chitosan thiol modifier is prepared by adding thiol glycolic acid and carbodiimide hydrochloride to a chitosan solution in a mass ratio of chitosan:thiol glycolic acid:carbodiimide hydrochloride = (1-1.5):(0.5-0.6):(0.3-0.4), and then reacting at 40-45°C under nitrogen protection, followed by acetone precipitation, centrifugation, washing, dialysis and freeze-drying.

[0015] In the above drug, the preparation method of the trimethyl chitosan-fucoidan complex includes: preparing 1.0mg / mL-1.5mg / mL trimethyl chitosan solution and 1.0mg / mL-1.5mg / mL fucoidan solution; under stirring, adding the trimethyl chitosan solution into the fucoidan solution in a volume ratio of trimethyl chitosan solution:fucoidan solution = 1:(0.4-0.6), and placing in an ice water bath, ultrasonic treatment to obtain a mixture A; adding sodium tripolyphosphate aqueous solution to the mixture A, stirring for 30-50 minutes to obtain a mixture B, centrifugation, washing with ultrapure water, and freeze-drying to obtain the trimethyl chitosan-fucoidan complex.

[0016] In the above preparation method of the trimethyl chitosan-fucoidan complex, 1.0wt%-1.2wt% acetic acid aqueous solution is used to prepare 1.0mg / mL-1.5mg / mL trimethyl chitosan solution; ultrapure water is used to prepare 1.0mg / mL-1.5mg / mL fucoidan solution; the ultrasonic treatment is at 200W-250W for 5-8 minutes; the amount of the added sodium tripolyphosphate aqueous solution is 1%-3% of the volume of the mixture A; the concentration of the sodium tripolyphosphate aqueous solution is 1.0wt%-1.5wt%; the stirring speed is 400rpm-500rpm; the centrifugation is at 4-6°C and 8000rpm-10000rpm for 15-20 minutes; and the ultrapure water washing is performed for 2-3 times.

[0017] In the above drug, the preparation method of the high-adhesion biomimetic carrier comprises: preparing 4.0wt%-5.0wt% of a silk fibroin solution, 2.0wt%-3.0wt% of a hyaluronic acid-thiol derivative solution, 2.0wt%-2.5wt% of a chitosan thiol modifier solution, and 2.0wt%-2.5wt% of a pectin solution; the volume ratio of the silk fibroin solution: the hyaluronic acid-thiol derivative solution: the chitosan thiol modifier solution: the pectin solution is (3-4):(2-2.5):(2-2.5):(3-4); the silk fibroin solution and the pectin solution are stirred uniformly first, and then the hyaluronic acid-thiol derivative solution and the chitosan thiol modifier solution are added in sequence under continuous stirring; after uniform stirring, the pH is adjusted to 6.0-6.5, and continuous stirring is performed for 2h-3h to obtain the high-adhesion biomimetic carrier.

[0018] In the above preparation method of the high-adhesion biomimetic carrier, the silk fibroin solution with a concentration of 4.0wt%-5.0wt% is prepared by using 40℃-45℃ ultrapure water; the hyaluronic acid-thiol derivative solution with a concentration of 2.0wt%-3.0wt% is prepared by using a phosphate buffer with a pH of 5.5-5.8; the chitosan thiol modifier solution with a concentration of 2.0wt%-2.5wt% is prepared by using a 0.1M-0.12M hydrochloric acid aqueous solution; and the pectin solution with a concentration of 2.0wt%-2.5wt% is prepared by using 45℃-50℃ ultrapure water.

[0019] In the above preparation method of the high-adhesion biomimetic carrier, the stirring speed is 200rpm-300rpm; and the pH is adjusted by using a 0.1M-0.12M sodium hydroxide aqueous solution.

[0020] In the above preparation method of the high-adhesion biomimetic carrier, the preparation method of the chitosan thiol modifier comprises: preparing a chitosan solution with a concentration of 1.0g / 100mL-1.5g / 100mL; adding thiol glycolic acid and carbodiimide hydrochloride into the chitosan solution according to the mass ratio of chitosan:thiol glycolic acid:carbodiimide hydrochloride=(1-1.5):(0.5-0.6):(0.3-0.4); stirring and reacting at 40℃-45℃ for 24h-30h under nitrogen protection to obtain a reaction solution; adding acetone into the reaction solution and stirring for 10min-15min; standing and aging, centrifugation, washing the precipitate with an acetone aqueous solution, then diluting with ultrapure water, loading into a dialysis bag, dialyzing with ultrapure water for 48h-72h, and freeze-drying to obtain the chitosan thiol modifier.

[0021] In the preparation method of the above thiol-modified chitosan, a chitosan solution with a concentration of 1.0 g / 100 mL to 1.5 g / 100 mL is prepared by using 0.1 M to 0.15 M hydrochloric acid aqueous solution; the addition amount of the acetone is 3 times to 5 times the volume of the reaction solution; the acetone is pre-cooled acetone at 0°C to 4°C; the stirring speed is 200 rpm to 300 rpm; the standing and aging is at 10°C below for 40 min to 60 min; the centrifugation is at 8000 rpm to 10000 rpm below 10°C for 15 min to 20 min; the volume concentration of the acetone aqueous solution is 75% to 85%; the acetone aqueous solution is pre-cooled acetone aqueous solution at 0°C to 4°C; the washing times of the acetone aqueous solution are 2 to 3 times; the dilution with ultrapure water is to a concentration of 1.0 wt% to 3.0 wt%; the molecular weight cut-off of the dialysis bag is 6 kDa to 8 kDa; and the dialysis solution is replaced every 4 h to 8 h.

[0022] The preparation method of the above active pharmaceutical composition for treating oral ulcer includes the following steps:

[0023] S1: under the condition of ice water bath, poloxamer 407 is added to a phosphate buffer with a pH of 6.8 to 7.2 according to the formula amount under stirring until completely dissolved to form a base solution;

[0024] S2: under the condition of ice water bath and stirring, high-adhesion biomimetic carrier, trimethyl chitosan-fucoidan complex, glycerol and ascorbyl palmitate are sequentially added and stirred uniformly;

[0025] S3: the stirring speed is reduced, and lactoferrin peptide, spilanthes acuta flavone, brazilian bignay extract, isomaltooligosaccharide and menthol-β-cyclodextrin inclusion compound are sequentially added and mixed uniformly;

[0026] S4: standing to obtain the medicine.

[0027] In S1 of the above preparation method, the stirring speed is 150 rpm to 200 rpm.

[0028] In S2 of the above preparation method, the stirring speed is 150 rpm to 200 rpm.

[0029] In S3 of the above preparation method, the stirring speed is reduced to 60 rpm to 80 rpm.

[0030] In S3 of the above preparation method, after mixing uniformly, the pH is adjusted to 6.8 to 7.2.

[0031] In S4 of the above preparation method, the standing is at 4°C to 6°C for 12 h to 16 h.

[0032] Use of the above-mentioned medicine in the preparation of a medicine for treating oral ulcer diseases.

[0033] The active pharmaceutical composition for treating oral ulcer provided by the present application has the beneficial effects including:

[0034] Firstly, the medicine of the present application solves the core pain points of traditional oral ulcer medicines, such as short-acting, large side effects and easy recurrence, through the synergistic system of complex treatment, long-acting adhesion, safe regulation and microecological prevention. The specific principles are as follows: (1) covering the pathological link: the active ingredient combination is aimed at bacterial infection, inflammatory response and mucosal repair, forming a closed loop treatment of anti-infection, inflammation and promoting healing, matching the pathological mechanism of oral ulcer infection, inflammation and epithelial apoptosis. (2) Long-acting adhesion to prolong drug efficacy: through the multiple adhesion mechanisms of electrostatic crosslinking, hydrogen bond and covalent bond of the composite carrier, the drug loss caused by saliva flushing is reduced, the limitation of traditional medicines completely lost within 2 hours is broken through, and the frequency of drug use is reduced. (3) Safe low toxicity and wide population adaptation: the component ratio is optimized to avoid the problems of hemolysis and osmotic pressure caused by excessive active ingredients, low-stimulus excipients are selected to avoid the side effects of chlorhexidine staining and hormone atrophy, and the medicine is suitable for sensitive groups such as children and pregnant women.

[0035] Secondly, the active ingredients construct a complex treatment core: lactoferrin peptide contains hydrophobic amino acid residues, which can destroy the cell membrane of pathogenic bacteria and regulate the immune response of the body to help inhibit the spread of infection. The polyphenolic components of spilanthes acuta flavonoids can scavenge inflammation-related active oxygen, reduce the release of inflammatory factors such as TNF-α and IL-6, and relieve ulcer pain. Its antioxidant properties can also protect other active ingredients from being oxidized and inactivated. Brazilian Bego extract: contains active polypeptides, which can specifically bind to the epidermal growth factor receptor on the surface of human epithelial cells, activate the cell proliferation signaling pathway, and accelerate the repair and regeneration of mucosal epithelial cells.

[0036] Three, functional adjuvant realizes long-acting, controlled release, comfort: (1) trimethyl chitosan-fucoidan complex: positive and negative charge polysaccharide forms a dense nanogel network by electrostatic crosslinking, and wraps the active ingredients to avoid premature release; the amino and hydroxyl groups on the surface of the complex form hydrogen bonds with the carboxyl and hydroxyl groups of the mucosal glycoprotein, thereby enhancing the mucosal adhesion. (2) High adhesion biomimetic carrier (silk fibroin + hyaluronic acid-thiol derivative + chitosan thiol modifier + pectin): silk fibroin provides film-forming mechanical strength to form a continuous protective film covering the ulcer surface; chitosan thiol modifier enhances adhesion while assisting in antibiosis; pectin assists adhesion through hydrogen bonds, and the four work together to form a firm, biocompatible adhesive film, thereby prolonging the drug retention time. (3) Poloxamer 407: the warm-sensitive property facilitates drug administration, and after contacting the wound, the drug is quickly solidified and locked, while providing a cooling sensation to relieve pain. (4) Ascorbyl palmitate: antioxidant, scavenges free radicals in the system, protecting active ingredients from oxidative degradation. (5) Glycerol: humectant, maintains a moist environment on the ulcer surface, which is conducive to epithelial cell migration and repair, and prevents the drug film from drying out and falling off. (6) Menthol-β-cyclodextrin inclusion complex: inclusion technology masks the bitter taste and releases menthol to provide a cooling sensation, improving the drug experience. (7) Phosphate buffer: simulates the physiological pH of the oral cavity, reducing the irritation of the drug to the ulcer wound.

[0037] Four, in the trimethyl chitosan-fucoidan complex, the volume ratio of trimethyl chitosan to fucoidan is 1: (0.4-0.6), which balances the positive charge of trimethyl chitosan with the negative charge of fucoidan to form a neutral gel; the complex at this ratio can bind to the mucosa through hydrogen bonds, reducing the loss of saliva flushing, and providing microcarrier support for long-acting drug effects.

[0038] Five, in the drug preparation method: ice water bath inhibits the premature gelation of poloxamer 407, ensuring uniform mixing of adjuvants and matrix, and avoiding local concentration unevenness; high-speed stirring ensures that the carrier, complex and other high molecular adjuvants are fully dissolved and dispersed to form a uniform matrix; finally, low-speed stirring avoids the destruction of the molecular structure of lactoferrin peptide and Brazilian Bego extract at high rotation speed, protecting their biological activity; low-temperature standing naturally eliminates system bubbles, avoiding bubbles in the drug film leading to poor adhesion, and low temperature reduces the degradation of active ingredients, ensuring the stability of the drug system. DETAILED DESCRIPTION

[0039] The application will be further described below in conjunction with specific implementation examples, but the application is not limited to these examples.

[0040] Example 1

[0041] An active pharmaceutical composition for treating oral ulcer is prepared from the following raw materials in mass percentage: 2.0% lactoferrin peptide, 1.5% purslane flavone, 1.2% Brazilian propolis extract, 6.5% trimethyl chitosan-fucoidan complex, 10.0% high-adhesion biomimetic carrier, 19.0% poloxamer 407, 5.0% oligoisomaltose, 0.6% menthol-beta-cyclodextrin inclusion compound, 0.3% ascorbyl palmitate, 4.5% glycerol, and the balance is phosphate buffer solution with pH 7.0, and the sum of the mass percentages of the above components is 100%.

[0042] The trimethyl chitosan-fucoidan complex is prepared by the following method: a trimethyl chitosan solution with a concentration of 1.2 mg / mL is prepared by using 1.1 wt% acetic acid aqueous solution; a fucoidan solution with a concentration of 1.2 mg / mL is prepared by using ultrapure water; under the condition of stirring at 450 rpm, the trimethyl chitosan solution is slowly added to the fucoidan solution at a volume ratio of 1:0.5, the mixture is placed in an ice water bath, and ultrasonic treatment is performed at 200 W for 6 min to obtain a mixture A; 2% of the volume of the mixture A is added with a 1.3 wt% sodium tripolyphosphate aqueous solution, and stirring is performed at 450 rpm for 40 min to obtain a mixture B; the mixture B is centrifuged at 5°C and 9000 rpm for 18 min, the precipitate is washed twice with ultrapure water, and freeze-drying is performed to obtain the trimethyl chitosan-fucoidan complex.

[0043] The high-adhesion biomimetic carrier is a quaternary composite matrix composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier, and pectin. The high-adhesion biomimetic carrier is prepared by the following method: a silk fibroin solution with a concentration of 4.5 wt% is prepared by using ultrapure water at 42°C; a hyaluronic acid-thiol derivative solution with a concentration of 2.5 wt% is prepared by using a phosphate buffer solution with pH 5.6; a chitosan thiol modifier solution with a concentration of 2.3 wt% is prepared by using a 0.11M hydrochloric acid aqueous solution; a pectin solution with a concentration of 2.2 wt% is prepared by using ultrapure water at 48°C; the silk fibroin solution and the pectin solution are first stirred uniformly at 250 rpm, and then the hyaluronic acid-thiol derivative solution and the chitosan thiol modifier solution are sequentially added under continuous stirring, and after uniform stirring, the pH is adjusted to 6.2 by using a 0.11M sodium hydroxide aqueous solution, and continuous stirring is performed at 250 rpm for 2.5 h to obtain the high-adhesion biomimetic carrier.

[0044] The preparation method of the chitosan thiol modifier comprises the following steps: preparing a chitosan solution with a concentration of 1.3 g / 100 mL by using 0.12 M hydrochloric acid aqueous solution; adding thiolactic acid and carbodiimide hydrochloride into the chitosan solution according to the mass ratio of chitosan:thiolactic acid:carbodiimide hydrochloride = 1.2:0.55:0.35; stirring and reacting for 26 h under the conditions of nitrogen protection, 42 DEG C and 250 rpm; adding 4 times the volume of 2 DEG C pre-cooled acetone into the reaction solution, stirring for 12 min under 250 rpm, standing and aging for 50 min at 6 DEG C, centrifuging for 18 min under the conditions of 6 DEG C and 9000 rpm, and taking the precipitate; washing the precipitate twice with 2 DEG C pre-cooled acetone water solution with a volume concentration of 80%, and then diluting to a concentration of 2.0 wt% with ultrapure water, loading into a dialysis bag with a molecular weight cut-off of 6 kDa, dialyzing for 60 h with ultrapure water as the dialysis liquid, replacing the dialysis liquid every 6 h, and finally freeze-drying to obtain the chitosan thiol modifier.

[0045] The preparation method of the menthol-beta-cyclodextrin inclusion compound comprises the following steps: preparing a saturated aqueous solution of beta-cyclodextrin at 65 DEG C according to the mass ratio of beta-cyclodextrin to menthol = 1:0.13; preparing a menthol solution with a concentration of 1 g / 6 mL by using anhydrous ethanol; adding the menthol solution into the beta-cyclodextrin saturated solution under stirring at 350 rpm, cooling to room temperature after stirring at 65 DEG C and 350 rpm for 2.5 h, crystallizing in a 5 DEG C refrigerator, collecting the precipitate by suction filtration, washing with 5 DEG C pure water, vacuum drying at 42 DEG C, crushing, and passing through a 200-mesh sieve to obtain the menthol-beta-cyclodextrin inclusion compound.

[0046] The preparation method of the active pharmaceutical composition for treating oral ulcer comprises the following steps:

[0047] S1: under the condition of ice water bath, adding poloxamer 407 into a phosphate buffer solution with pH 7.0 according to the formula amount under continuous stirring at 180 rpm until complete dissolution to form a base solution;

[0048] S2: under the condition of ice water bath and continuous stirring at 180 rpm, sequentially adding the high-adhesion biomimetic carrier, the trimethyl chitosan-fucoidan complex, glycerol and ascorbyl palmitate, and stirring uniformly;

[0049] S3: reducing the stirring speed to 70 rpm, sequentially adding the lactoferrin peptide, the spilanthes acmella flavone, the brazilian propolis extract, the isomalto-oligosaccharide and the menthol-beta-cyclodextrin inclusion compound, and mixing uniformly; adjusting the pH of the system to 7.0;

[0050] S4: standing at 5 DEG C for 14 h to naturally eliminate air bubbles to form a stable drug system.

[0051] Example 2

[0052] An active pharmaceutical composition for treating oral ulcer is prepared from the following raw materials in mass percentage: 1.0% lactoferrin peptide, 2.0% purslane flavone, 1.0% Brazilian bignay extract, 8.0% trimethyl chitosan-fucoidan complex, 8.0% high-adhesion biomimetic carrier, 20.0% poloxamer 407, 3.0% oligoisomaltose, 1.0% menthol-β-cyclodextrin inclusion compound, 0.1% ascorbyl palmitate, 5.0% glycerol, and the balance being a phosphate buffer solution at pH 6.8, with the sum of the mass percentages of the above components being 100%.

[0053] The trimethyl chitosan-fucoidan complex is prepared by the following method: a trimethyl chitosan solution with a concentration of 1.5 mg / mL is prepared using 1.0 wt% acetic acid aqueous solution; a fucoidan solution with a concentration of 1.0 mg / mL is prepared using ultrapure water; under stirring at 500 rpm, the trimethyl chitosan solution is slowly added to the fucoidan solution at a volume ratio of 1:0.4, and the mixture is placed in an ice water bath and treated with ultrasonic waves at 250 W for 5 min to obtain a mixture A; 1% of the mixture A is added with 1.0 wt% sodium tripolyphosphate aqueous solution, and stirred at 500 rpm for 30 min to obtain a mixture B; the mixture B is centrifuged at 6℃ and 8000 rpm for 20 min, and the precipitate is washed twice with ultrapure water and freeze-dried to obtain the trimethyl chitosan-fucoidan complex.

[0054] The high-adhesion biomimetic carrier is a quaternary composite matrix composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier, and pectin. The high-adhesion biomimetic carrier is prepared by the following method: a silk fibroin solution with a concentration of 4.0 wt% is prepared using 45℃ ultrapure water; a hyaluronic acid-thiol derivative solution with a concentration of 2.0 wt% is prepared using a phosphate buffer solution at pH 5.8; a chitosan thiol modifier solution with a concentration of 2.0 wt% is prepared using 0.12M hydrochloric acid aqueous solution; a pectin solution with a concentration of 2.0 wt% is prepared using 50℃ ultrapure water; the silk fibroin solution and the pectin solution are first stirred uniformly at 300 rpm, and then the hyaluronic acid-thiol derivative solution and the chitosan thiol modifier solution are sequentially added under continuous stirring, and after uniform stirring, the pH is adjusted to 6.5 using 0.1M sodium hydroxide aqueous solution, and the mixture is continuously stirred at 200 rpm for 3h to obtain the high-adhesion biomimetic carrier.

[0055] The preparation method of the chitosan thiol modifier comprises the following steps: preparing a chitosan solution with a concentration of 1.5 g / 100 mL by using 0.1M hydrochloric acid aqueous solution; adding thiolactic acid and carbodiimide hydrochloride into the chitosan solution according to the mass ratio of chitosan:thiolactic acid:carbodiimide hydrochloride = 1:0.6:0.3; stirring and reacting for 30 hours under the conditions of nitrogen protection, 45 DEG C and 200 rpm to obtain a reaction solution; adding 3 times the volume of 4 DEG C pre-cooled acetone into the reaction solution, stirring for 15 minutes under 200 rpm, standing and aging for 60 minutes at 4 DEG C, centrifuging for 15 minutes under the conditions of 4 DEG C and 10000 rpm, and taking the precipitate; washing the precipitate with 75% pre-cooled acetone aqueous solution for 3 times, then diluting to a concentration of 1.0 wt% with ultrapure water, loading into a dialysis bag with a molecular weight cut-off of 8 kDa, dialyzing for 48 hours with ultrapure water as the dialysis liquid, replacing the dialysis liquid every 8 hours, and finally freeze-drying to obtain the chitosan thiol modifier.

[0056] The preparation method of the menthol-beta-cyclodextrin inclusion compound comprises the following steps: preparing a saturated aqueous solution of beta-cyclodextrin at 70 DEG C to obtain a beta-cyclodextrin saturated solution, and preparing a menthol solution with a concentration of 1 g / 5 mL by using anhydrous ethanol; adding the menthol solution into the beta-cyclodextrin saturated solution under stirring at 400 rpm, cooling to room temperature after stirring at 60 DEG C and 400 rpm for 2 hours, crystallizing in a 6 DEG C refrigerator, collecting the precipitate by suction filtration, washing with 4 DEG C purified water, vacuum drying at 45 DEG C, crushing, and passing through a 200 mesh sieve to obtain the menthol-beta-cyclodextrin inclusion compound.

[0057] The preparation method of the active pharmaceutical composition for treating oral ulcer comprises the following steps:

[0058] S1: adding poloxamer 407 into a phosphate buffer solution with pH 6.8 according to the formula amount under the condition of ice water bath and continuous stirring at 150 rpm until completely dissolved to form a base solution;

[0059] S2: sequentially adding the high-adhesion biomimetic carrier, the trimethyl chitosan-fucoidan complex, glycerol and ascorbyl palmitate under the condition of ice water bath and continuous stirring at 150 rpm until uniformly stirred;

[0060] S3: sequentially adding the lactoferrin peptide, the spilanthes acmella flavone, the brazilin extract, the isomaltooligosaccharide and the menthol-beta-cyclodextrin inclusion compound under the condition of reducing the stirring speed to 80 rpm until uniformly mixed, and adjusting the pH of the system to 6.8;

[0061] S4: standing at 4 DEG C for 16 hours to naturally eliminate air bubbles to form a stable drug system.

[0062] Example 3

[0063] An active pharmaceutical composition for treating oral ulcer is prepared from the following raw materials in mass percentage: 3.0% lactoferrin peptide, 1.0% purslane flavone, 1.5% Brazilian propolis extract, 5.0% trimethyl chitosan-fucoidan complex, 12.0% high-adhesion biomimetic carrier, 18.0% poloxamer 407, 7.0% oligoisomaltose, 0.3% menthol-beta-cyclodextrin inclusion compound, 0.5% ascorbyl palmitate, 2.0% glycerol, and the balance being a phosphate buffer solution with pH 7.2, with the sum of the mass percentages of the components being 100%.

[0064] The trimethyl chitosan-fucoidan complex is prepared by the following method: a trimethyl chitosan solution with a concentration of 1.0 mg / mL is prepared using 1.2 wt% acetic acid aqueous solution; a fucoidan solution with a concentration of 1.5 mg / mL is prepared using ultrapure water; under stirring at 400 rpm, the trimethyl chitosan solution is slowly added to the fucoidan solution at a volume ratio of 1:0.6, and the mixture is placed in an ice water bath and treated with ultrasonic waves at 200 W for 8 min to obtain a mixture A; 3% of the mixture A is added with 1.5 wt% sodium tripolyphosphate aqueous solution, and stirred at 400 rpm for 50 min to obtain a mixture B; the mixture B is centrifuged at 4°C and 10000 rpm for 15 min, and the precipitate is washed with ultrapure water for 3 times and freeze-dried to obtain the trimethyl chitosan-fucoidan complex.

[0065] The high-adhesion biomimetic carrier is a quaternary composite matrix composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier and pectin. The high-adhesion biomimetic carrier is prepared by the following method: a silk fibroin solution with a concentration of 5.0 wt% is prepared using 40°C ultrapure water; a hyaluronic acid-thiol derivative solution with a concentration of 3.0 wt% is prepared using a phosphate buffer solution with pH 5.5; a chitosan thiol modifier solution with a concentration of 2.5 wt% is prepared using 0.1M hydrochloric acid aqueous solution; a pectin solution with a concentration of 2.5 wt% is prepared using 45°C ultrapure water; the silk fibroin solution and the pectin solution are first stirred uniformly at 200 rpm, and then the hyaluronic acid-thiol derivative solution and the chitosan thiol modifier solution are added in sequence under continuous stirring, and after uniform stirring, the pH is adjusted to 6.0 using 0.12M sodium hydroxide aqueous solution, and the mixture is continuously stirred at 300 rpm for 2 h to obtain the high-adhesion biomimetic carrier.

[0066] The preparation method of the chitosan thiol modifier comprises the following steps: preparing a chitosan solution with a concentration of 1.0 g / 100 mL by using 0.15 M hydrochloric acid aqueous solution; adding thiolactic acid and carbodiimide hydrochloride into the chitosan solution according to the mass ratio of chitosan:thiolactic acid:carbodiimide hydrochloride = 1.5:0.5:0.4; stirring and reacting for 24 h under the conditions of nitrogen protection, 40 DEG C and 300 rpm; adding 5 times the volume of pre-cooled acetone at 0 DEG C into the reaction solution, stirring for 10 min at 300 rpm, standing and aging for 60 min at 8 DEG C, centrifuging for 20 min at 8 DEG C and 8000 rpm, and taking the precipitate; washing the precipitate twice with pre-cooled acetone aqueous solution with a volume concentration of 85 DEG C at 0 DEG C, then diluting to a concentration of 3.0 wt% with ultrapure water, loading into a dialysis bag with a molecular weight cut-off of 6 kDa, dialyzing for 72 h with ultrapure water as the dialysis liquid, replacing the dialysis liquid every 4 h, and finally freeze-drying to obtain the chitosan thiol modifier.

[0067] The preparation method of the menthol-beta-cyclodextrin inclusion compound comprises the following steps: preparing a saturated aqueous solution of beta-cyclodextrin at 60 DEG C according to the mass ratio of beta-cyclodextrin to menthol = 1:0.15; preparing a menthol solution with a concentration of 1 g / 8 mL by using anhydrous ethanol; adding the menthol solution into the beta-cyclodextrin saturated solution under stirring at 300 rpm, stirring for 3 h at 70 DEG C, then cooling to room temperature, crystallizing by cold storage at 4 DEG C, collecting the precipitate by suction filtration, washing with purified water at 6 DEG C, vacuum drying at 40 DEG C, crushing, and passing through a 200-mesh sieve to obtain the menthol-beta-cyclodextrin inclusion compound.

[0068] The preparation method of the active pharmaceutical composition for treating oral ulcer comprises the following steps:

[0069] S1: under the condition of ice water bath, adding poloxamer 407 into a phosphate buffer solution with pH 7.2 according to the formula amount under continuous stirring at 200 rpm until completely dissolved to form a base solution;

[0070] S2: under the condition of ice water bath and continuous stirring at 200 rpm, sequentially adding the high-adhesion biomimetic carrier, the trimethyl chitosan-fucoidan complex, glycerol and ascorbyl palmitate, and stirring uniformly;

[0071] S3: reducing the stirring speed to 60 rpm, sequentially adding lactoferrin peptide, spilanthes acuta flavone, brazilian propolis extract, isomaltooligosaccharide and menthol-beta-cyclodextrin inclusion compound, and mixing uniformly; adjusting the pH of the system to 7.2;

[0072] S4: standing at 6 DEG C for 12 h to naturally eliminate air bubbles to form a stable drug system.

[0073] The raw material sources of the above-mentioned each embodiment medicine are as follows: lactoferrin peptide is from Xi'an Juntai Biological Technology Co., Ltd., model JTSWRTDB-0. Spilanthes acuta flavone is from Baoji Liupan Yun Biological Technology Co., Ltd., purity 80%. Brazilian bixiga extract is from Shaanxi Guan Chen Biological Technology Co., Ltd., model GC-BXBG-01. Poloxamer 407 is from Shanxi Jinyang Pharmaceutical Auxiliary Material Co., Ltd. Oligoisomaltose is from Guangzhou Pangu Chemical Product Co., Ltd. Ascorbyl palmitate is from Nanjing Xintai Biological Technology Co., Ltd. Glycerol is from Nanjing Yingguan New Material Technology Co., Ltd., medical grade glycerol. Menthol is from Jiangxi Hairui Natural Plant Co., Ltd. β-Cyclodextrin is from Hebei Chuangzhiyuan Biological Technology Co., Ltd. Trimethyl chitosan is from Shanghai Yingyibing Biological Technology Co., Ltd., N-trimethyl chitosan. Fucus vesiculosus polysaccharide is from Xi'an Ousai Biological Technology Co., Ltd., specification 20:1. Sodium tripolyphosphate is from Shandong Suan Chemical Co., Ltd., food grade. Silk fibroin is from Hubei Hongxinruiyu Fine Chemical Co., Ltd., water-soluble. Hyaluronic acid-thiol derivative is from Beijing Jingpi Technology Co., Ltd., model JPHA-3. Pectin is from Guangdong Qingwen Biological Technology Co., Ltd. Chitosan is from Shenzhen Yibo Hui Biological Technology Co., Ltd., chitosan (acid-soluble). Thioglycolic acid is from Beijing Nai Zhen Biochemical Technology Co., Ltd. Carbodiimide hydrochloride is from Wuhan Huaxiang Kejebioengineering Co., Ltd., EDC hydrochloride.

[0074] Comparative Example 1

[0075] The difference from Example 1 is that spilanthes acuta flavone is modified to 2.2% and Brazilian bixiga extract is modified to 0.5%.

[0076] Comparative Example 2

[0077] The difference from Example 1 is that spilanthes acuta flavone is modified to 0.5% and Brazilian bixiga extract is modified to 2.2%.

[0078] Comparative Example 3

[0079] The difference from Example 1 is that trimethyl chitosan-fucus vesiculosus polysaccharide compound is modified to 2%.

[0080] Comparative Example 4

[0081] The difference from Example 1 is that trimethyl chitosan-fucus vesiculosus polysaccharide compound is replaced by trimethyl chitosan.

[0082] Comparative Example 5

[0083] The difference from Example 1 is that trimethyl chitosan-fucus vesiculosus polysaccharide compound is directly replaced by trimethyl chitosan powder and fucus vesiculosus polysaccharide powder in proportion.

[0084] Comparative Example 6

[0085] The difference from Example 1 is that in the preparation of the trimethyl chitosan-fucoidan complex, the volume ratio of the trimethyl chitosan solution and the fucoidan solution is modified to = 1:1.5.

[0086] Comparative Example 7

[0087] The difference from Example 1 is that the high-adhesion biomimetic carrier is modified to 5%.

[0088] Comparative Example 8

[0089] The difference from Example 1 is that in the preparation method of the high-adhesion biomimetic carrier, hyaluronic acid-thiol derivative is replaced by hyaluronic acid (same manufacturer, model JPHA-1).

[0090] Comparative Example 9

[0091] The difference from Example 1 is that in the preparation method of the high-adhesion biomimetic carrier, chitosan thiol modifier is replaced by chitosan.

[0092] Comparative Example 10

[0093] The difference from Example 1 is that in the preparation method of the high-adhesion biomimetic carrier, hyaluronic acid-thiol derivative is replaced by hyaluronic acid, and chitosan thiol modifier is replaced by chitosan.

[0094] I. Hemolysis test:

[0095] (1) Purpose of detection:

[0096] To evaluate the hemolysis and safety of the drug composition.

[0097] (2) Samples and grouping:

[0098] Cell strain: human red blood cells.

[0099] Sample preparation: each example and comparative example 1 to comparative example 2 drug without poloxamer 407, and the phosphate buffer in the drug stock solution is replaced by culture medium, all the samples to be tested are sterilized by ultraviolet, and three concentrations of samples are set: concentrated to 1 / 2 volume concentration V, original volume concentration V, and diluted to 2 volumes concentration V2. Grouping settings: 1 / 2

[0100] Blank control group: only containing culture medium (used to calibrate the instrument).

[0101] Negative control group: 0.5 mL red blood cell suspension + 1 mL PBS (pH 7.4).

[0102] Positive control group: 0.5 mL red blood cell suspension + 1 mL distilled water. ​

[0103] Experimental group: 1 mL sample + 0.5 mL red blood cell suspension.

[0104] (3) Detection steps and parameters:

[0105] Take the anticoagulated blood (EDTA anticoagulation) of healthy people, wash it with normal saline for 3 times (each time at 4°C, centrifuge at 1500 rpm for 10 min), take the red blood cells and prepare a 2% (v / v) suspension with normal saline. After 2h in a 37°C water bath, centrifuge at 4°C, 1500 rpm for 10 min, take the supernatant, and measure the absorbance value A at 540 nm. Subtract the background value of the blank control group. Hemolysis rate%= (A experimental group-A negative control) / (A positive control-A negative control) x 100%. Take the average value of 6 replicate wells.

[0106] II. Antimicrobial activity test:

[0107] (1) Purpose: Quickly and intuitively compare the inhibition ability of each sample on common oral pathogenic bacteria.

[0108] (2) Strains: Staphylococcus aureus (ATCC25923), oral streptococcus (ATCC35668 Streptococcus mutans), Candida albicans (ATCC10231).

[0109] (3) Samples: Each example and comparative example 1 to comparative example 2 drug stock solution without poloxamer 407; negative control (PBS); positive control (0.2% chlorhexidine for bacteria, 0.1% clotrimazole for fungi).

[0110] (4) Steps: Adjust the activated strain to 0.5 McFarland turbidity (1.5 x 10 8 CFU / mL) with normal saline. Dip the bacterial suspension with a sterile cotton swab and evenly coat it on the surface of the corresponding agar plate (BHI for bacteria, SDA for fungi), and let it stand for 5 min to allow the culture medium to absorb the moisture. Use a sterile puncher to punch holes on the agar (diameter 6 mm), and add 60 μL of the sample to be tested to each hole. After 2h of upright culture in a 37°C constant temperature incubator, the bacterial plate is inverted and cultured for another 22h; the Candida albicans plate is cultured at 28°C for 48h. Measure the diameter (mm) of the inhibition zone with a vernier caliper, and take the average value of 3 parallel samples.

[0111] III. Anti-inflammatory activity detection:

[0112] (1) Purpose of detection:

[0113] To evaluate the inhibitory effect of the pharmaceutical composition on the inflammatory response of macrophages and explore its anti-inflammatory mechanism.

[0114] (2) Samples and grouping:

[0115] Cell line: mouse monocyte macrophage leukemia cell (RAW264.7).

[0116] Test sample: each of the examples and the drug stock solution of Comparative Example 1 to Comparative Example 2 not containing poloxamer 407.

[0117] Stimulant: lipopolysaccharide (LPS), working concentration 1 μg / mL.

[0118] Grouping:

[0119] Blank group: cells + complete culture medium;

[0120] Model group (LPS group): cells + LPS stimulation;

[0121] Dosing group: cells + drug (pre-treated for 2 h) + LPS stimulation.

[0122] (3) Detection steps and indexes:

[0123] Cell inoculation and treatment: RAW264.7 cells were inoculated in a 24-well plate at a density of 5 × 10 5 cells / well, and cultured for 24 h. Pre-treatment and stimulation were performed according to the grouping, and each group had 3 replicate wells. ELISA detection of inflammatory factors: after 24 h of stimulation, the cell supernatant was collected. According to the operation instruction of the ELISA kit, the content of TNF-α and IL-6 (pg / mL) in the supernatant was detected.

[0124] Four, detection of promotion of epithelial cell proliferation:

[0125] (1) Purpose of detection:

[0126] The proliferation ability of the drug composition on human immortalized keratinocytes (HaCaT) was quantitatively evaluated, so as to verify the efficacy of promoting oral mucosal ulcer healing.

[0127] (2) Cell line: human immortalized keratinocytes (HaCaT).

[0128] (3) Test sample preparation and grouping: drug stock solution of each of the examples and Comparative Example 1 to Comparative Example 2 not containing poloxamer 407.

[0129] (4) Experimental grouping:

[0130] Blank control group: only containing serum-free culture medium (no cells, used for calibrating the enzyme marker).

[0131] Negative control group: cells + serum-free culture medium.

[0132] Experimental group: cells + serum-free culture medium containing the corresponding working solution of the sample to be tested (each example and each comparative example).

[0133] (5) Detection steps and parameters:

[0134] Cell seeding: HaCaT cells in logarithmic growth phase were digested with 0.25% Trypsin-EDTA, resuspended in complete culture medium, and counted. The cell suspension was seeded into 96-well plates at a density of 5 × 10⁶ cells / well (100 μL per well). 3 Cells per well. Pre-culture the culture plate in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere completely.

[0135] Drug administration: Carefully aspirate the old culture medium from each well. According to the groups, add 100 μL of pre-prepared serum-free culture medium containing the drug to each well. Each sample group and control group has 6 replicates (n=6).

[0136] Culture and Detection: After adding the reagent, the culture plate was returned to the incubator for further incubation. Detection was performed 48 hours after incubation. Add 10 μL of LCK-8 reagent to each well. Gently shake the culture plate to mix. Return the culture plate to the incubator and incubate for another 2 hours. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader.

[0137] Data processing: Calculate the mean OD value for each group (excluding the OD value of the blank group). Cell proliferation rate (%) = (OD experimental group / OD negative control group) × 100%.

[0138] V. Mucosal adhesion force test:

[0139] (1) Objective: To evaluate the intrinsic adhesion properties of drugs to mucosa by simulating the oral saliva flushing environment under the condition of eliminating the interference of temperature-sensitive gel matrix.

[0140] (2) Samples: Drug stock solutions of each example and comparative examples 3 to 10 without poloxamer 407.

[0141] (3) Procedure: Fix the porcine mucosa (1cm × 1cm) in the device. Take 0.2g of sample (calculate the initial content of purslane brass W1) and spread it evenly on the mucosa surface. Turn on the constant flow pump and continuously rinse with artificial saliva at 37℃ at a flow rate of 2mL / min. Stop rinsing at 0.5h, 1h, and 2h respectively, remove the mucosa, and thoroughly elute the residual drug with an appropriate amount of PBS. Determine the content of purslane brass W2 in the eluent by HPLC. Calculate the purslane brass retention rate % = (W2 / W1) × 100%. 5 parallel samples.

[0142] Table 1. Test Results (Average)

[0143]

[0144] Table 2 Test Results (Continued)

[0145]

[0146] From the above results, the drugs of Examples 1 to 3 achieve a comprehensive balance of safety, antibacterial and anti-inflammatory, sustained release adhesion and healing promotion by optimizing the component ratio and complex structure.

[0147] Comparative Example 1 (spironolactone 2.2%, Brazilian Beggiaea extract 0.5%): Spironolactone, as a polyphenolic antibacterial and anti-inflammatory ingredient, relies on uniform dispersion in an aqueous solution for its effect. When the concentration is increased from 1.5% in Example 1 to 2.2%, it is easy to form aggregates with lactoferrin peptides in the system, which also have a hydrophobic structure, through hydrophobic interaction. This aggregate reduces the contact area of spironolactone with bacterial cell membranes and inflammatory cells. Brazilian Beggiaea extract contains active polypeptides that activate the epidermal growth factor receptor on the surface of HaCaT cells, promote protein phosphorylation, and then start the cell proliferation cycle. When the concentration of the extract is reduced from 1.2% to 0.5%, the polypeptide content is insufficient to effectively bind, resulting in blocked proliferation signaling and a decrease in the proliferation rate of epithelial cells. Excessive spironolactone molecules insert into the phospholipid bilayer of the red blood cell membrane, slightly damaging the membrane's fluidity and integrity, resulting in a small amount of hemoglobin leakage and a slight increase in hemolysis rate.

[0148] Comparative Example 2 (spironolactone 0.5%, Brazilian Beggiaea extract 2.2%): When the concentration of spironolactone is reduced to 0.5%, the number of molecules is insufficient to achieve an effective antibacterial concentration: for Staphylococcus aureus, oral Streptococcus, and other gram-positive bacteria, it cannot increase membrane permeability by damaging the lipoteichoic acid structure on the cell membrane; for Candida albicans, it also cannot effectively inhibit the synthesis of chitin in the cell wall. At the same time, low concentrations of flavonoids cannot effectively scavenge reactive oxygen species (ROS) produced by RAW264.7 cells after activation by LPS, and the accumulation of ROS will further activate the NF-κB pathway, leading to a significant increase in the release of TNF-α and IL-6. When the concentration of Brazilian Beggiaea extract is increased to 2.2%, the osmotic pressure of the system increases (polypeptide components are one of the main contributors to osmotic pressure), and red blood cells will undergo dehydration and shrinkage in a high-osmotic environment, with decreased membrane stability; in addition, excessive polypeptides will non-specifically bind to receptors on the surface of HaCaT cells, competing with EGFR binding sites and inhibiting normal proliferation signaling, resulting in a decrease in the proliferation rate of epithelial cells.

[0149] Comparative Example 3 (trimethyl chitosan-fucoidan complex 2%): Trimethyl chitosan (positively charged) and fucoidan (sulfate group-containing, negatively charged) form a nanogel network through electrostatic interaction, and the concentration directly determines the compactness of the network structure. The 6.5% complex concentration in Example 1 forms a dense network that can effectively encapsulate active ingredients such as purslane flavonoids; when the concentration is reduced to 2%, the network pore size expands, and the connection force between the network nodes weakens. The amino groups (-NH2) and hydroxyl groups (-OH) on the surface of the complex form hydrogen bonds with the carboxyl groups (-COOH) and hydroxyl groups on the surface of the oral mucosa epithelial cells. Reducing the concentration reduces the number of hydrogen bond binding sites per unit area, and the binding strength of the complex to the mucosa decreases. Under the continuous flushing of artificial saliva, the drug is easily detached from the mucosal surface, and the mucosal retention rate is greatly reduced.

[0150] Comparative Example 4 (trimethyl chitosan instead of complex): Trimethyl chitosan alone can only form a loose linear aggregation structure through intermolecular van der Waals forces and hydrogen bonds, lacking the strong electrostatic crosslinking between the sulfate groups of fucoidan and the quaternary ammonium groups, and cannot form a stable three-dimensional gel network. This loose structure is easily dissociated in the release liquid, resulting in a large amount of drug release in a short time. The binding of trimethyl chitosan alone to the mucosa relies only on the hydrogen bonding between the amino groups and the glycoproteins, and the binding force is much weaker than that of the complex gel, resulting in a decrease in the mucosal retention rate.

[0151] Comparative Example 5 (trimethyl chitosan powder + fucoidan powder instead of complex): In the preparation process of the complex in Example 1, ultrasonic treatment allows the two polysaccharide molecules to fully disperse and form a nanoscale mixed system, and the subsequent addition of sodium tripolyphosphate further promotes crosslinking to form a dense gel; while the direct mixing of powders has not been subjected to ultrasonic and crosslinking treatment, and is only a macroscopic physical mixture, the polysaccharide molecules have not been fully contacted, and the electrostatic interaction is limited to the surface of the powder, and a uniform nanogel network cannot be formed.

[0152] Comparative Example 6 (complex preparation volume ratio 1:1.5): In Example 1, when the volume ratio of trimethyl chitosan to fucoidan is 1:0.5, the ratio of positive and negative charges is close to 1:1, forming a neutral complex gel, the system has the highest viscosity, and the network structure is the most stable; when the volume ratio changes to 1:1.5, the excess of fucoidan leads to an excess of negative charges, making it difficult for polysaccharide molecules to form tight crosslinking due to electrostatic repulsion. Excess fucoidan exists in the gel network in the form of free chains, which hinders the encapsulation of active ingredients and promotes the diffusion of drug molecules in the release liquid, resulting in accelerated drug release. At the same time, the excess of negative charges in the complex produces electrostatic repulsion with the mucosal epithelial cells also carrying negative charges, weakening the binding force and reducing the mucosal retention rate.

[0153] Comparative Example 7 (high adhesion biomimetic carrier 5%): The high adhesion biomimetic carrier is composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier and pectin, and its adhesion performance depends on the formation of a continuous film structure: silk fibroin provides the mechanical strength of the film, hyaluronic acid-thiol derivative and chitosan thiol modifier bind to the mucosa through disulfide bonds of thiol groups, and pectin assists adhesion through hydrogen bonds. When the concentration decreases from 10.0% to 5%, the carrier cannot form a continuous and complete film on the mucosa surface, and the performance decreases. The discontinuity of the film leads to the exposure of the drug to the saliva flushing environment, and the insufficient number of thiol groups (the concentration of thiol groups is positively correlated with the concentration of the carrier), and the reduction of disulfide bond binding sites with the mucosa, which have a dual effect of greatly reducing the mucosal retention rate; in addition, the discontinuous film structure cannot effectively hinder the diffusion of the drug, resulting in accelerated drug release.

[0154] Comparative Example 8 (hyaluronic acid instead of hyaluronic acid-thiol derivative): Hyaluronic acid-thiol derivative binds to the cystine residues in the keratin of the oral mucosa epithelial cell surface through thiol groups. After being replaced by ordinary hyaluronic acid, the binding of the carrier to the mucosa only depends on hydrogen bonds and van der Waals forces, and under the continuous flushing of artificial saliva, the hydrogen bonds are easily broken, resulting in a decrease in the mucosal retention rate.

[0155] Comparative Example 9 (chitosan instead of chitosan thiol modifier): Chitosan thiol modifier can form a more stable three-dimensional network structure with the thiol groups of hyaluronic acid-thiol derivative, enhancing the mechanical strength and drug wrapping capacity of the carrier. Ordinary chitosan has no thiol groups, and the network structure of the carrier is relatively loose. The binding of ordinary chitosan to the mucosa only depends on the hydrogen bond interaction between the amino groups and glycoproteins, resulting in a decrease in the mucosal retention rate.

[0156] Comparative Example 10 (hyaluronic acid + chitosan instead of two thiol derivatives): After the absence of hyaluronic acid-thiol derivative and chitosan thiol modifier, the binding of the carrier to the mucosa only depends on the hydrogen bond interaction of ordinary hyaluronic acid and chitosan, and the binding force is extremely weak, which is easily detached under the flushing of saliva, and the mucosal retention rate greatly decreases.

Claims

1. An active pharmaceutical composition for treating oral ulcers, characterized in that, The drug is made from the following raw materials in the following mass percentages: 1.0%–3.0% lactoferrin peptide, 1.0%–2.0% purslane flavonoids, 1.0%–1.5% Brazilian bigot extract, 5.0%–8.0% trimethyl chitosan-fucoidan polysaccharide complex, 8.0%–12.0% highly adhesive biomimetic carrier, 18.0%–20.0% poloxamer 407, 3.0%–7.0% isomaltooligosaccharide, 0.3%–1.0% menthol-β-cyclodextrin inclusion complex, 0.1%–0.5% ascorbate palmitate, 2.0%–5.0% glycerol, with the balance being phosphate buffer at pH 6.8–7.

2. The sum of the mass percentages of the above components is 100%. The trimethyl chitosan-fucus vesiculosus polysaccharide complex was prepared by mixing a trimethyl chitosan solution with a concentration of 1.0 mg / mL to 1.5 mg / mL and a fucus vesiculosus polysaccharide solution with a concentration of 1.0 mg / mL to 1.5 mg / mL at a volume ratio of 1:(0.4 to 0.6), followed by ultrasonication in an ice-water bath, then adding an aqueous solution of sodium tripolyphosphate, stirring, centrifuging to collect the precipitate, washing, and freeze-drying. The highly adhesive biomimetic carrier is a quaternary composite matrix composed of silk fibroin, hyaluronic acid-thiol derivative, chitosan thiol modifier, and pectin; the highly adhesive biomimetic carrier is prepared by mixing a solution of 4.0wt%–5.0wt% silk fibroin, a solution of 2.0wt%–3.0wt% hyaluronic acid-thiol derivative, a solution of 2.0wt%–2.5wt% chitosan thiol modifier, and a solution of 2.0wt%–2.5wt% pectin in a volume ratio of (3–4):(2–2.5):(2–2.5):(3–4) and stirring at pH 6.0–6.5 for 2–3 hours. The chitosan thiol modifier is prepared by adding thioglycolic acid and carbodiimide hydrochloride to a chitosan solution in a mass ratio of chitosan:thioglycolic acid:carbodiimide hydrochloride = (1-1.5):(0.5-0.6):(0.3-0.4), reacting the mixture at 40-45°C under nitrogen protection, followed by acetone precipitation, centrifugation, washing, dialysis, and freeze-drying. The lactoferrin peptide is sourced from Xi'an Juntian Biotechnology Co., Ltd., model JTSWRTDB-0; The Brazilian Bigo extract was sourced from Shaanxi Guanchen Biotechnology Co., Ltd., model number GC-BXBG-01. The hyaluronic acid-thiol derivative is from Beijing Jingbi Technology Co., Ltd., model JPHA-3.

2. The active pharmaceutical composition for treating oral ulcers according to claim 1, characterized in that, The preparation method of the trimethyl chitosan-fucus vesiculosus polysaccharide complex includes: preparing a trimethyl chitosan solution with a concentration of 1.0 mg / mL to 1.5 mg / mL and a fucus vesiculosus polysaccharide solution with a concentration of 1.0 mg / mL to 1.5 mg / mL; adding the trimethyl chitosan solution to the fucus vesiculosus polysaccharide solution at a volume ratio of 1:(0.4 to 0.6) under stirring, placing the mixture in an ice-water bath, and sonicating to obtain mixture A; adding sodium tripolyphosphate aqueous solution to mixture A, stirring for 30 min to 50 min to obtain mixture B, centrifuging, collecting the precipitate, washing with ultrapure water, and freeze-drying to obtain the trimethyl chitosan-fucus vesiculosus polysaccharide complex.

3. The active pharmaceutical composition for treating oral ulcers according to claim 2, characterized in that, A trimethyl chitosan solution with a concentration of 1.0 mg / mL to 1.5 mg / mL was prepared using a 1.0 wt% to 1.2 wt% aqueous acetic acid solution; a Fucus vesiculosus polysaccharide solution with a concentration of 1.0 mg / mL to 1.5 mg / mL was prepared using ultrapure water; the sonication was performed at 200 W to 250 W for 5 min to 8 min; the amount of sodium tripolyphosphate aqueous solution added was 1% to 3% of the volume of mixture A; the concentration of the sodium tripolyphosphate aqueous solution was 1.0 wt% to 1.5 wt%; the stirring speed was 400 rpm to 500 rpm; the centrifugation was performed at 4℃ to 6℃ and 8000 rpm to 10000 rpm for 15 min to 20 min; the ultrapure water washing was performed 2 to 3 times.

4. The active pharmaceutical composition for treating oral ulcers according to claim 1, characterized in that, The preparation method of the highly adhesive biomimetic carrier includes: preparing a 4.0wt%–5.0wt% silk fibroin solution, a 2.0wt%–3.0wt% hyaluronic acid-thiol derivative solution, a 2.0wt%–2.5wt% chitosan thiol modified solution, and a 2.0wt%–2.5wt% pectin solution; the volume ratio of silk fibroin solution: hyaluronic acid-thiol derivative solution: chitosan thiol modified solution: pectin solution = (3–4): (2–2.5): (2–2.5): (3–4); first, the silk fibroin solution and pectin solution are stirred evenly; then, the hyaluronic acid-thiol derivative solution and chitosan thiol modified solution are added sequentially under continuous stirring; after stirring evenly, the pH is adjusted to 6.0–6.5, and stirring is continued for 2–3 hours to obtain the highly adhesive biomimetic carrier.

5. The active pharmaceutical composition for treating oral ulcers according to claim 4, characterized in that, Prepare a silk fibroin solution with a concentration of 4.0wt% to 5.0wt% using ultrapure water at 40℃ to 45℃; prepare a hyaluronic acid-thiol derivative solution with a concentration of 2.0wt% to 3.0wt% using phosphate buffer solution at pH 5.5 to 5.8; prepare a chitosan thiol modified solution with a concentration of 2.0wt% to 2.5wt% using 0.1M to 0.12M hydrochloric acid aqueous solution; and prepare a pectin solution with a concentration of 2.0wt% to 2.5wt% using ultrapure water at 45℃ to 50℃.

6. The active pharmaceutical composition for treating oral ulcers according to claim 4, characterized in that, The stirring speed is 200 rpm to 300 rpm; the pH is adjusted using a 0.1 M to 0.12 M sodium hydroxide aqueous solution.

7. The active pharmaceutical composition for treating oral ulcers according to claim 4, characterized in that, The preparation method of the chitosan thiol modified compound includes: preparing a chitosan solution of 1.0 g / 100 mL to 1.5 g / 100 mL; adding thioglycolic acid and carbodiimide hydrochloride to the chitosan solution at a mass ratio of chitosan:thioglycolic acid:carbodiimide hydrochloride = (1-1.5):(0.5-0.6):(0.3-0.4), stirring and reacting at 40℃-45℃ for 24-30 h under nitrogen protection to obtain a reaction solution; adding acetone to the reaction solution, stirring for 10-15 min, allowing it to stand for aging, centrifuging, taking the precipitate, washing it with acetone aqueous solution, then diluting it with ultrapure water, placing it in a dialysis bag, dialyzing with ultrapure water as the dialysate for 48-72 h, and freeze-drying to obtain the chitosan thiol modified compound.

8. The active pharmaceutical composition for treating oral ulcers according to claim 7, characterized in that, A chitosan solution with a concentration of 1.0 g / 100 mL to 1.5 g / 100 mL is prepared using a 0.1 M to 0.15 M hydrochloric acid aqueous solution; the amount of acetone added is 3 to 5 times the volume of the reaction solution; the acetone is pre-cooled at 0℃ to 4℃; the stirring speed is 200 rpm to 300 rpm; the aging process is carried out at below 10℃ for 40 min to 60 min; the centrifugation is carried out at below 10℃ and 8000 rpm for 15 min to 20 min; the volume concentration of the acetone aqueous solution is 75% to 85%; the acetone aqueous solution is pre-cooled at 0℃ to 4℃; the acetone aqueous solution is washed 2 to 3 times; the ultrapure water is diluted to a concentration of 1.0 wt% to 3.0 wt%; the molecular weight cutoff of the dialysis bag is 6 kDa to 8 kDa; the dialysis solution is replaced every 4 h to 8 h.

9. A method for preparing an active pharmaceutical composition for treating oral ulcers according to claim 1, characterized in that, Includes the following steps: S1: Under ice-water bath conditions, add poloxamer 407 to a phosphate buffer solution with pH 6.8–7.2 according to the formula amount, and stir until completely dissolved to form a matrix solution; S2: Under ice-water bath and stirring, add the high-adhesion biomimetic carrier, trimethyl chitosan-fucoidan polysaccharide complex, glycerol and ascorbate palmitate in sequence, and stir until uniform; S3: Reduce the stirring speed and add lactoferrin peptide, purslane flavonoids, Brazilian bigo extract, isomaltooligosaccharide and menthol-β-cyclodextrin inclusion complex in sequence, and mix well; S4: Let stand to obtain the drug.

10. A method for preparing an active pharmaceutical composition for treating oral ulcers according to claim 9, characterized in that, In S1, the stirring speed is 150 rpm to 200 rpm; in S2, the stirring speed is 150 rpm to 200 rpm; in S3, the stirring speed is reduced to 60 rpm to 80 rpm; in S3, after mixing evenly, the pH is adjusted to 6.8 to 7.2; in S4, the standing is carried out at 4℃ to 6℃ for 12h to 16h.

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