Multifunctional pharmaceutical composition for comprehensive tooth repair and preparation method thereof

Abstract

The present application relates to a kind of multifunctional pharmaceutical composition of dental comprehensive repair and its preparation method, belong to biopharmaceutical technical field, and pharmaceutical composition includes keratin peptide, coral fungus extract, eggshell derived nano calcium phosphate, catechin zinc complex, hydroxyapatite, modified chitosan-PEG gel and other components.Keratin peptide is the product between 3kDa-10kDa obtained by keratinase enzymolysis to white goose velvet;Coral fungus extract is the product obtained by cellulase, subtilisin and bromelain enzymolysis to coral fungus, and purified by G-25 dextran gel column;Modified chitosan-PEG gel is the product obtained by sodium cyanoborohydride stabilization and glycerol plasticization to the reactant of chitosan and dialdehyde polyethylene glycol.Integrated multiple functions such as antibacterial, mineralization and tissue repair, through optimizing component and dosage form, realize efficient, minimally invasive dental damage repair, and good biocompatibility and long-acting.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a multifunctional pharmaceutical composition for comprehensive dental restoration and its preparation method. Background Technology

[0002] Tooth damage mainly stems from two main scenarios: extrinsic factors, such as tooth decay, dental trauma, and wear; and intrinsic factors, such as pulp necrosis and periodontal disease. Tooth damage not only reduces quality of life but can also lead to complications such as pulpitis and periapical periodontitis. Therefore, the development of dental restorative drugs remains a core focus in the field of oral healthcare.

[0003] From the perspective of tooth physiology and repair mechanisms, damage to different parts requires targeted intervention: enamel, as the outermost protective layer of the tooth, has no cellular structure and cannot regenerate on its own; once damaged, it can only be filled with exogenous materials. Dentin contains dentin cell processes, and after damage, it can achieve limited regeneration by forming reparative dentin, but the regeneration speed is slow and the repair range is limited. Although dental pulp tissue has a certain self-repair ability, it is prone to necrosis after infection or severe damage, requiring medication to promote the recovery of pulp vitality. Based on this, the development of dental restorative drugs has undergone an iteration from local symptomatic treatment to targeted repair: early treatments focused on symptom relief, such as fluoride preparations that prevent caries progression by promoting enamel remineralization, and eugenol used for pulp soothing and analgesia; subsequently, targeted regenerative drugs emerged, such as preparations containing mineralizing components that can repair early caries lesions, and drugs containing growth factors that can induce dentin regeneration. These drugs have become commonly used clinical methods for mild tooth damage.

[0004] However, existing dental restorative medications have significant limitations and are insufficient to meet the comprehensive restorative needs of complex dental damage. Specific problems include:

[0005] (1) Single function, unable to cover multiple types of damage. Existing drugs are mostly targeted at a single repair scenario. For example, fluoride drugs can only prevent tooth decay and cannot repair dentin defects; growth factor drugs can promote dentin regeneration but lack antibacterial ability and cannot deal with infectious damage. Clinically, multiple drugs need to be used in combination, which increases the complexity of the treatment process.

[0006] (2) Insufficient biocompatibility and long-term effectiveness. Some restorative drugs have poor biocompatibility with tooth tissues, which can easily lead to marginal leakage and secondary caries; some biological agents have poor stability and are easily degraded in the moist oral environment, requiring frequent administration to maintain the effect, resulting in low patient compliance.

[0007] To address the aforementioned issues, the development of multifunctional pharmaceutical compositions for comprehensive dental restoration has become an inevitable trend. These compositions need to integrate multiple functions: first, possessing both antibacterial and mineralizing capabilities to simultaneously address infection and defects; second, incorporating tissue repair components to adapt to complex injury scenarios; and third, improving drug stability and longevity through optimized dosage forms to reduce the frequency of administration. Ultimately, this will achieve a one-stop solution to the multidimensional needs of dental damage repair, driving oral healthcare towards greater efficiency and minimally invasive procedures. Summary of the Invention

[0008] To address the shortcomings of existing dental restorative drugs, such as limited functionality, insufficient biocompatibility, and poor long-term effectiveness, this invention provides a multifunctional pharmaceutical composition for comprehensive dental restoration and its preparation method. The composition is formulated with ingredients including keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, hydroxyapatite, and modified chitosan-PEG gel. It integrates multiple functions such as antibacterial, mineralization, and tissue repair. By optimizing the composition and dosage form, it achieves highly efficient and minimally invasive dental damage repair with good biocompatibility and long-term effectiveness. The specific technical solution is as follows:

[0009] A multifunctional pharmaceutical composition for comprehensive dental restoration comprises the following raw materials in parts by weight: 5-8 parts keratin peptide, 3-5 parts coral fungus extract, 8-12 parts eggshell-derived nano-calcium phosphate, 2-3 parts zinc catechin complex, 10-15 parts hydroxyapatite, 60-80 parts modified chitosan-PEG gel, and deionized water, pH 6.5-7.5;

[0010] The keratin peptides are products between 3kDa and 10kDa obtained by enzymatic hydrolysis of white goose down using keratinase; the coral fungus extract is a product obtained by enzymatic hydrolysis of coral fungus using cellulase, subtilisin, and bromelain, followed by purification using a G-25 dextran gel column; the eggshell-derived nano-calcium phosphate is obtained by dissolving goose eggshell powder in an aqueous acetic acid solution to form a calcium acetate solution, reacting it with an aqueous NaOH solution to generate calcium hydroxide precipitate, and then reacting it with disodium hydrogen phosphate to generate eggshell-derived nano-calcium phosphate; the catechin zinc complex is a catechin zinc complex generated by the reaction of catechin and zinc sulfate; the modified chitosan-PEG gel is a product obtained by reacting chitosan with dialdehyde polyethylene glycol, stabilizing it with sodium cyanoborohydride, and plasticizing it with glycerol.

[0011] The preparation method of the above-mentioned keratin peptides includes: soaking white goose down in NaOH aqueous solution, filtering, washing, drying, pulverizing into powder, adding 8 to 12 times the mass of deionized water to adjust the pH to 8.0 to 9.0, adding keratinase, enzymatically hydrolyzing at 50℃ to 55℃ for 2.5 to 3.5 hours, inactivating the enzyme, centrifuging, taking the supernatant, ultrafiltration membrane ultrafiltration, taking the fraction between 3 kDa and 10 kDa, freeze-drying to obtain keratin peptides.

[0012] In the above method for preparing keratin peptides, the concentration of the NaOH aqueous solution is 0.5 mol / L to 1.0 mol / L; the soaking is performed at 50℃ to 70℃ for 2 to 3 hours; the washing is performed with deionized water until the pH reaches 6.5 to 7.0; the amount of keratinase added is 2% to 3% of the powder mass; the enzyme inactivation is performed at 90℃ to 95℃ for 10 to 15 minutes; and the centrifugation is performed at 6000 rpm to 8000 rpm for 10 to 20 minutes.

[0013] The preparation method of the above-mentioned coral fungus extract includes: pulverizing coral fungus into fungal powder, adding it to deionized water, adjusting the pH to 4.5-5.5, adding cellulase, and enzymatically hydrolyzing at 50℃-55℃ for 1.5-2 hours, adjusting the pH to 6.5-7.5, adding subtilisin and bromelain, and enzymatically hydrolyzing at 50℃-55℃ for 2-2.5 hours, inactivating the enzymes, centrifuging, collecting the supernatant, concentrating under reduced pressure, loading the sample onto a G-25 dextran gel column, rinsing with deionized water to remove impurities, eluting with ammonium acetate buffer, collecting the eluent, concentrating under reduced pressure, and freeze-drying to obtain the coral fungus extract.

[0014] In the above method for preparing coral fungus extract, the coral fungus is a dried coral fungus fruiting body; the particle size of the fungal powder is sieved through an 80-100 mesh sieve; the amount of deionized water used is 8 to 12 times the mass of the fungal powder; the amounts of cellulase, subtilisin, and bromelain added are all 1% to 1.5% of the mass of the fungal powder; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 15 minutes, followed by cooling to room temperature; the centrifugation is performed at 6000 rpm to 8000 rpm for 10 to 20 minutes.

[0015] The preparation method of the above-mentioned eggshell-derived nano-calcium phosphate includes: crushing goose eggshells into eggshell powder, adding the eggshell powder to a 1M-2M acetic acid aqueous solution, stirring, filtering to remove insoluble matter, obtaining a calcium acetate solution, adjusting the pH to 11.0-12.0 with a 1M-2M NaOH aqueous solution to generate calcium hydroxide precipitate, washing with deionized water, resuspending with deionized water to obtain a calcium hydroxide suspension, adding a 0.1M-1M disodium hydrogen phosphate aqueous solution, controlling the calcium-phosphorus molar ratio to (1.5-1.7):1, adjusting the pH to 9.0-10.5 with a 0.1M-1M NaOH aqueous solution, stirring the reaction, centrifuging, washing the precipitate with deionized water to pH 6.5-7.5, drying, and dry ball milling to a particle size D50 of 50nm-150nm to obtain eggshell-derived nano-calcium phosphate.

[0016] The preparation method of the above-mentioned zinc catechin complex includes: preparing a 5wt% to 8wt% catechin solution with deionized water, and preparing a 0.1M to 0.2M zinc sulfate solution with deionized water; adding the zinc sulfate solution to the catechin solution according to the mass ratio of zinc element in catechin to zinc sulfate of 1:(0.18 to 0.25), adjusting the pH to 7.0 to 7.5, stirring the reaction in the dark, adding anhydrous ethanol, allowing it to stand to precipitate, centrifuging, washing the precipitate with a mixed solution of anhydrous ethanol and acetone, and vacuum drying to obtain the zinc catechin complex.

[0017] The preparation method of the modified chitosan-PEG gel includes: preparing a chitosan solution with a concentration of 3wt% to 5wt% using a 1wt% to 2wt% acetic acid solution; adding dialdehyde polyethylene glycol at a mass ratio of chitosan: dialdehyde polyethylene glycol = 1:(0.5 to 0.8); adjusting the pH to 6.0 to 6.5 with NaOH aqueous solution; stirring the reaction to obtain a reaction solution; adding sodium cyanoborohydride to continue the reaction; adding anhydrous ethanol; allowing the mixture to stand to precipitate; filtering; washing the precipitate with anhydrous ethanol; and drying to obtain modified chitosan; preparing a modified chitosan solution with a concentration of 2wt% to 5wt% using deionized water; adding glycerol with a final concentration of 0.5wt% to 2wt%; stirring; and adjusting the viscosity to 150 Pa·s to 250 Pa·s to obtain the modified chitosan-PEG gel.

[0018] In the above method for preparing modified chitosan-PEG gel, the NaOH aqueous solution is 0.8M to 1M NaOH aqueous solution; the stirring reaction is carried out at 50℃ to 70℃ and 100rpm to 200rpm for 4h to 8h; and the amount of anhydrous ethanol is 2 to 3 times the volume of the reaction liquid.

[0019] The preparation method of the above-mentioned multifunctional pharmaceutical composition for comprehensive dental restoration includes the following steps:

[0020] Keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, hydroxyapatite, and modified chitosan-PEG gel are mixed according to the mass fractions, and 10 to 20 parts of deionized water are added. The mixture is stirred evenly at 20°C to 35°C, and the pH is adjusted to 6.5 to 7.5 to form a gel, thus obtaining the pharmaceutical composition.

[0021] The use of the above-mentioned pharmaceutical composition in the preparation of dental restorative drugs.

[0022] This invention provides a multifunctional pharmaceutical composition for comprehensive dental restoration and its preparation method, with the following beneficial effects:

[0023] I. The pharmaceutical composition of this invention integrates three core functions—antibacterial, mineralization, and tissue repair—to simultaneously address infection control and tissue regeneration in dental injuries. Utilizing a gel formulation, it is suitable for various complex injury scenarios, including dental caries, dentin defects, and pulp infections. Its long-lasting and stable release mechanism reduces the frequency of administration, lowering the treatment burden on patients. Furthermore, its composition is highly compatible with the physiological structure of teeth, achieving repair while minimizing additional damage to dental tissues, thus promoting the development of oral healthcare towards minimally invasive and precise procedures.

[0024] Second, the polar groups such as carboxyl and hydroxyl groups on the molecular chain of keratin peptides can capture calcium ions and phosphate ions, providing nucleation sites for the deposition of minerals such as hydroxyapatite, guiding the orderly growth of crystals, and forming a dense repair layer; the molecular weight range of 3kDa to 10kDa balances permeability and stability, the low molecular weight segment can penetrate dentinal tubules to exert a deep repair effect, and the high molecular weight segment can maintain structural stability, avoid being rapidly degraded by the oral environment, and achieve a long-lasting repair effect.

[0025] Third, the coral fungus extract contains polysaccharides and glycoproteins that can mimic the organic matrix of natural teeth. It not only binds mineralized ions and regulates the local microenvironment, promoting the transformation of amorphous minerals into crystalline states, but also enhances the inhibitory effect of antibacterial components on cariogenic bacteria. The synergistic enzymatic hydrolysis of three proteases precisely hydrolyzes coral fungus proteins, generating bioactive peptides with specific structures and functions. Using a single enzyme or lacking any one enzyme will result in insufficient types and amounts of bioactive peptides. G-25 dextran gel adsorption and ammonium acetate buffer elution specifically retain water-soluble active ingredients, avoiding the residue of small molecule impurities, while maintaining the spatial stability of the bioactive peptides.

[0026] IV. Eggshell-derived nano-calcium phosphate, as a highly efficient mineralization enhancer, has a high specific surface area due to its nano-sized particles, which can quickly release calcium and phosphate ions and form a synergistic mineralization system with hydroxyapatite. Hydroxyapatite provides the basic mineralization framework, while nano-calcium phosphate accelerates the ion deposition rate and significantly improves the remineralization efficiency of tooth enamel. In addition, eggshell-derived nano-calcium phosphate has good biocompatibility.

[0027] The reaction conditions of calcium-phosphorus molar ratio (1.5-1.7):1 and pH 9.0-10.5 ensure the formation of calcium phosphate crystals consistent with the mineral composition of teeth, avoiding the generation of impurity phases; dry ball milling controls the particle size D50 to 50nm-150nm, ensuring that the particles can penetrate the dentinal tubules and achieve deep mineralization.

[0028] V. Catechin Zinc Complex (Prepared by Coordination Reaction of Catechin and Zinc Sulfate): Through coordination bonds, synergistic functions are achieved. Zinc ions enhance the anti-degradation ability of catechins, preventing them from rapidly oxidizing and becoming ineffective in the moist environment of the oral cavity. At the same time, zinc ions themselves can destroy bacterial cell membranes, supplementing the antibacterial effect. Compared with catechins alone, it significantly improves the antibacterial rate against Streptococcus mutans and Porphyromonas gingivalis, and the antibacterial effect is more lasting.

[0029] VI. Modified Chitosan-PEG Gel (Chitosan reacts with dialdehyde polyethylene glycol, stabilized by sodium cyanoborohydride): The bioadhesive properties of chitosan allow the gel to adhere tightly to the tooth surface, preventing it from falling off during chewing and saliva rinsing; PEG modification enhances the gel's flexibility and biocompatibility, reducing irritation to gingival tissue; sodium cyanoborohydride reduces the Schiff base formed by chitosan and dialdehyde polyethylene glycol to a stable CN bond, preventing the gel from disintegrating in a humid environment and ensuring slow and continuous drug release.

[0030] VII. Hydroxyapatite serves as the basic framework for mineralization. Its composition is consistent with the main mineral components of tooth enamel and dentin, exhibiting excellent biocompatibility. It can act as a skeleton for mineral deposition, synergistically constructing a stable repair mineralization layer with eggshell-derived nano-calcium phosphate, while simultaneously filling defects on the tooth surface and restoring tooth morphology.

[0031] 8. Synergistic effect of the formula, multiple components work together to overcome the functional limitations of single components: Catechin zinc complex, keratin peptides and coral fungus extract synergistically inhibit the growth of cariogenic bacteria and infectious bacteria, and remove infection interference in the mineralization process; Eggshell-derived nano-calcium phosphate and hydroxyapatite, guided by keratin peptides and coral fungus extract, quickly form a mineralization layer in the defect area. At the same time, the dense structure of the mineralization layer can further block bacterial invasion, forming a closed-loop protection of antibacterial, mineralization and barrier, and avoiding secondary caries.

[0032] 9. Synergistic effect of dosage form and ingredients to ensure long-lasting effect and biocompatibility: The adhesive and sustained-release properties of modified chitosan-PEG gel can lock active ingredients such as keratin peptides, coral fungus extract, and zinc catechin complex in the repair area, release them slowly, prolong the efficacy, and reduce the frequency of administration; at the same time, the biocompatibility of the gel encapsulates mineralized particles and antibacterial ingredients, avoiding direct contact with gingival tissue and preventing irritation, thus improving the safety of treatment. Detailed Implementation

[0033] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0034] Example 1

[0035] A multifunctional pharmaceutical composition for comprehensive dental restoration comprises the following raw materials in parts by weight: 6.5 parts keratin peptide, 4 parts coral fungus extract, 10 parts eggshell-derived nano-calcium phosphate, 2.5 parts zinc catechin complex, 13 parts hydroxyapatite, 70 parts modified chitosan-PEG gel, and deionized water.

[0036] The preparation method of keratin peptides includes: soaking white goose down (free from pathogen contamination) in 0.8 mol / L NaOH aqueous solution at 60℃ for 2.5 h; after filtration, washing with deionized water to pH 6.8, drying and pulverizing into powder, adding 10 times the mass of deionized water to adjust the pH to 8.5, adding 2.5% of the mass of keratinase, enzymatically hydrolyzing at 53℃ for 3 h, inactivating the enzyme at 92℃ for 13 min, centrifuging at 7000 rpm for 15 min, taking the supernatant, ultrafiltration membrane ultrafiltration, taking the fraction between 3 kDa and 10 kDa, freeze-drying to obtain keratin peptides.

[0037] The preparation method of coral fungus extract includes: taking dried coral fungus (Botrytis cinerea, a non-toxic edible fungus) fruiting bodies, crushing them through a 100-mesh sieve to obtain fungal powder, adding 10 times the mass of deionized water, adjusting the pH to 5.0, adding 1.2% of the fungal powder mass of cellulase, enzymatically hydrolyzing at 53℃ for 1.5h, adjusting the pH to 7.0, adding 1.3% of the fungal powder mass of Bacillus subtilis protease and 1.2% of the fungal powder mass of bromelain, enzymatically hydrolyzing at 53℃ for 2h, inactivating the enzyme at 88℃ for 13min; centrifuging at 7000rpm for 15min, collecting the supernatant, concentrating under reduced pressure at 48℃ to 25% of the volume, loading the sample onto a G-25 dextran gel column, rinsing with deionized water for 2.5 BV to remove impurities, then eluting with 0.08M ammonium acetate (NH4OAc) buffer (pH 6.9) for 4.5 BV, collecting the eluent, concentrating under reduced pressure at 48℃, and freeze-drying to obtain coral fungus extract.

[0038] The preparation method of eggshell-derived nano-calcium phosphate includes: washing and drying goose eggshells (removing the inner membrane and ensuring they are free of pathogen contamination), pulverizing them through a 100-mesh sieve to obtain eggshell powder; adding the eggshell powder to a 1.5M acetic acid aqueous solution at a mass ratio of 1:7, stirring at 25℃ for 1.5 hours, filtering to remove insoluble matter to obtain a calcium acetate solution, adjusting the pH to 11.5 with a 1.5M NaOH aqueous solution to generate calcium hydroxide precipitate, washing the precipitate twice with deionized water, resuspending it in 7 times its mass of deionized water to obtain a calcium hydroxide suspension, adding a 0.6M disodium hydrogen phosphate aqueous solution, controlling the calcium-to-phosphorus molar ratio to 1.6:1, and then using a 0.5M NaOH aqueous solution... The pH was adjusted to 9.5 with NaOH aqueous solution, and the mixture was stirred at 85℃ and 400 rpm for 3 h. After centrifugation at 9000 rpm for 15 min, the precipitate was washed with deionized water until the pH reached 7.0. It was then dried at 70℃ for 10 h and ball-milled until the particle size D50 was 118 nm, thus obtaining eggshell-derived nano-calcium phosphate.

[0039] The preparation method of the zinc catechin complex includes: preparing a 6.5 wt% catechin solution with deionized water and a 0.15 M zinc sulfate solution with deionized water; adding the zinc sulfate solution to the catechin solution according to the mass ratio of zinc element in catechin to zinc sulfate of 1:0.2, and adjusting the pH to 7.3 with 0.9 M NaOH aqueous solution; stirring the reaction at 50℃ and 300 rpm in the dark for 2 h, adding 2.5 times the volume of anhydrous ethanol to the reaction solution, and allowing it to stand at 3℃ for 3 h to precipitate; centrifuging at 7000 rpm for 15 min, washing the precipitate three times with a mixed solution of anhydrous ethanol and acetone (volume ratio 1.2:1), and vacuum drying at 50℃ for 10 h to obtain the zinc catechin complex.

[0040] The preparation method of modified chitosan-PEG gel includes: preparing a 4wt% chitosan solution using a 1.5wt% acetic acid solution; adding dialdehyde polyethylene glycol at a mass ratio of chitosan:dialdehyde polyethylene glycol = 1:0.65; adjusting the pH to 6.3 with 0.9M NaOH aqueous solution; stirring the mixture at 60℃ and 150rpm for 6 hours to obtain a reaction solution; adding 0.7% sodium cyanoborohydride (by mass of the reaction solution) as a reducing agent; and continuing the reaction under the same conditions. The Schiff base was stabilized for 1.5 h; then 2.5 times the volume of anhydrous ethanol was added to the reaction solution, and the mixture was allowed to stand to precipitate. The precipitate was filtered, washed twice with anhydrous ethanol, and dried under vacuum at 42 °C for 10 h to obtain modified chitosan (sodium cyanoborohydride residue was removed); a 3 wt% modified chitosan solution was prepared with deionized water, and glycerol was added to a final concentration of 1.2 wt%. The mixture was stirred at 25 °C and 120 rpm for 2 h, and the viscosity was adjusted to 200 Pa·s to obtain modified chitosan-PEG gel.

[0041] The preparation method of the above-mentioned multifunctional pharmaceutical composition for comprehensive dental restoration includes the following steps:

[0042] Keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, hydroxyapatite, and modified chitosan-PEG gel were mixed according to the mass fractions, and 15 parts of deionized water were added. The mixture was stirred evenly at 28°C, and the pH was adjusted to 7.0 to form a gel, thus obtaining the pharmaceutical composition.

[0043] Example 2

[0044] A multifunctional pharmaceutical composition for comprehensive dental restoration comprises the following raw materials in parts by weight: 5 parts keratin peptide, 5 parts coral fungus extract, 8 parts eggshell-derived nano-calcium phosphate, 3 parts zinc catechin complex, 10 parts hydroxyapatite, 80 parts modified chitosan-PEG gel, and deionized water.

[0045] The preparation method of keratin peptides includes: soaking white goose down (free from pathogen contamination) in 0.5 mol / L NaOH aqueous solution at 70℃ for 2 h; after filtration, washing with deionized water to pH 7.0, drying and pulverizing into powder, adding 8 times the mass of deionized water to adjust the pH to 9.0, adding 2% of the mass of keratinase, enzymatically hydrolyzing at 55℃ for 2.5 h, inactivating the enzyme at 95℃ for 10 min, centrifuging at 8000 rpm for 10 min, taking the supernatant, ultrafiltration membrane ultrafiltration, taking the fraction between 3 kDa and 10 kDa, freeze-drying to obtain keratin peptides.

[0046] The preparation method of coral fungus extract includes: taking dried coral fungus (Botrytis cinerea, a non-toxic edible fungus) fruiting bodies, crushing them through a 100-mesh sieve to obtain fungal powder, adding 8 times the mass of deionized water, adjusting the pH to 5.5, adding 1% of the fungal powder mass of cellulase, enzymatically hydrolyzing at 55℃ for 1.5h, adjusting the pH to 7.5, adding 1% of the fungal powder mass of Bacillus subtilis protease and 1.5% of the fungal powder mass of bromelain, enzymatically hydrolyzing at 50℃ for 2h, inactivating the enzyme at 85℃ for 15min; centrifuging at 6000rpm for 20min, collecting the supernatant, concentrating under reduced pressure at 45℃ to 30% of the volume, loading the sample onto a G-25 dextran gel column, rinsing with deionized water for 2BV to remove impurities, then eluting with 0.1M ammonium acetate (NH4OAc) buffer (pH 6.8) for 5BV, collecting the eluent, concentrating under reduced pressure at 45℃, and freeze-drying to obtain coral fungus extract.

[0047] The preparation method of eggshell-derived nano-calcium phosphate includes: washing and drying goose eggshells (removing the inner membrane and ensuring they are free of pathogen contamination), pulverizing them through a 120-mesh sieve to obtain eggshell powder; adding the eggshell powder to a 2M acetic acid aqueous solution at a mass ratio of 1:6, stirring at 20℃ for 2 hours, filtering to remove insoluble matter to obtain a calcium acetate solution, adjusting the pH to 12.0 with 1M NaOH aqueous solution to generate calcium hydroxide precipitate, washing the precipitate twice with deionized water, resuspending it with 8 times the mass of the precipitate in deionized water to obtain a calcium hydroxide suspension, adding 0.1M disodium hydrogen phosphate aqueous solution to control the calcium-to-phosphorus molar ratio to 1.7:1, adjusting the pH to 10.5 with 0.1M NaOH aqueous solution, stirring at 80℃ and 500rpm for 2 hours, centrifuging at 10000rpm for 10 minutes, washing the precipitate with deionized water to pH 7.5, drying at 60℃ for 12 hours, and dry ball milling to a particle size D50 of 50nm to obtain eggshell-derived nano-calcium phosphate.

[0048] The preparation method of zinc catechin complex includes: preparing an 8wt% catechin solution with deionized water and a 0.1M zinc sulfate solution with deionized water; adding the zinc sulfate solution to the catechin solution according to the mass ratio of zinc element in catechin to zinc sulfate of 1:0.25, and adjusting the pH to 7.5 with 0.8M NaOH aqueous solution; stirring the reaction at 40℃ and 400rpm in the dark for 1.5h to obtain a reaction solution; adding three times the volume of anhydrous ethanol to the reaction solution and allowing it to stand at 2℃ for 4h to precipitate; centrifuging at 6000rpm for 20min; washing the precipitate four times with a mixed solution of anhydrous ethanol and acetone (volume ratio 1:1); and vacuum drying at 40℃ for 12h to obtain zinc catechin complex.

[0049] The preparation method of modified chitosan-PEG gel includes: preparing a 5wt% chitosan solution with 1wt% acetic acid solution; adding dialdehyde polyethylene glycol at a mass ratio of chitosan:dialdehyde polyethylene glycol = 1:0.5; adjusting the pH to 6.0 with 1M NaOH aqueous solution; stirring at 70℃ and 100rpm for 8h to obtain a reaction solution; adding 0.5% sodium cyanoborohydride as a reducing agent and continuing the reaction for 2h under the same conditions to stabilize the Schiff base; then adding two volumes of anhydrous ethanol to the reaction solution; allowing the precipitate to stand and precipitate; filtering; washing the precipitate three times with anhydrous ethanol; and vacuum drying at 40℃ for 12h to obtain modified chitosan (with no sodium cyanoborohydride residue); preparing a 2wt% modified chitosan solution with deionized water; adding glycerol to a final concentration of 2wt%; stirring at 20℃ and 150rpm for 1h; and adjusting the viscosity to 150Pa·s to obtain modified chitosan-PEG gel.

[0050] The preparation method of the above-mentioned multifunctional pharmaceutical composition for comprehensive dental restoration includes the following steps:

[0051] Keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, hydroxyapatite, and modified chitosan-PEG gel were mixed according to the mass fractions, and 10 parts of deionized water were added. The mixture was stirred evenly at 35°C, and the pH was adjusted to 7.5 to form a gel, thus obtaining the pharmaceutical composition.

[0052] Example 3

[0053] A multifunctional pharmaceutical composition for comprehensive dental restoration comprises the following raw materials in parts by weight: 8 parts keratin peptide, 3 parts coral fungus extract, 12 parts eggshell-derived nano-calcium phosphate, 2 parts zinc catechin complex, 15 parts hydroxyapatite, 60 parts modified chitosan-PEG gel, and deionized water.

[0054] The preparation method of keratin peptides includes: soaking white goose down (free from pathogen contamination) in 1.0 mol / L NaOH aqueous solution at 50℃ for 3 h; after filtration, washing with deionized water to pH 6.5, drying and pulverizing into powder, adding 12 times the mass of deionized water to adjust the pH to 8.0, adding 3% of the mass of keratinase, enzymatically hydrolyzing at 50℃ for 3.5 h, inactivating the enzyme at 90℃ for 15 min, centrifuging at 6000 rpm for 20 min, taking the supernatant, ultrafiltration membrane ultrafiltration, taking the fraction between 3 kDa and 10 kDa, freeze-drying to obtain keratin peptides.

[0055] The preparation method of coral fungus extract includes: taking dried coral fungus (Botrytis cinerea, a non-toxic edible fungus) fruiting bodies, crushing them through an 80-mesh sieve to obtain fungal powder, adding 12 times the mass of deionized water, adjusting the pH to 4.5, adding 1.5% of the fungal powder mass of cellulase, enzymatically hydrolyzing at 50℃ for 2 hours, adjusting the pH to 6.5, adding 1.5% of the fungal powder mass of Bacillus subtilis protease and 1% of the fungal powder mass of bromelain, enzymatically hydrolyzing at 55℃ for 2.5 hours, inactivating the enzyme at 90℃ for 10 minutes; centrifuging at 8000 rpm for 10 minutes, collecting the supernatant, concentrating under reduced pressure at 50℃ to 20% of the volume, loading the sample onto a G-25 dextran gel column, rinsing with deionized water for 3 BV to remove impurities, then eluting with 0.05M ammonium acetate (NH4OAc) buffer (pH 7.0) for 4 BV, collecting the eluent, concentrating under reduced pressure at 50℃, and freeze-drying to obtain coral fungus extract.

[0056] The preparation method of eggshell-derived nano-calcium phosphate includes: washing and drying goose eggshells (removing the inner membrane and ensuring they are free of pathogen contamination), pulverizing them through an 80-mesh sieve to obtain eggshell powder; adding the eggshell powder to a 1M acetic acid aqueous solution at a mass ratio of 1:8, stirring at 30℃ for 1 hour, filtering to remove insoluble matter to obtain a calcium acetate solution, adjusting the pH to 11.0 with a 2M NaOH aqueous solution to generate calcium hydroxide precipitate, washing the precipitate three times with deionized water, resuspending it in 6 times the mass of the precipitate with deionized water to obtain a calcium hydroxide suspension, adding a 1M disodium hydrogen phosphate aqueous solution to control the calcium-to-phosphorus molar ratio to 1.5:1, adjusting the pH to 9.0 with a 1M NaOH aqueous solution, stirring at 90℃ and 300 rpm for 4 hours, centrifuging at 8000 rpm for 20 minutes, washing the precipitate with deionized water to pH 6.5, drying at 80℃ for 6 hours, and dry ball milling to a particle size D50 of 150 nm to obtain eggshell-derived nano-calcium phosphate.

[0057] The preparation method of zinc catechin complex includes: preparing a 5wt% catechin solution with deionized water and a 0.2M zinc sulfate solution with deionized water; adding the zinc sulfate solution to the catechin solution according to the mass ratio of zinc element in catechin to zinc sulfate of 1:0.18, and adjusting the pH to 7.0 with 1M NaOH aqueous solution; stirring the reaction at 60℃ and 200rpm in the dark for 3h to obtain a reaction solution; adding anhydrous ethanol twice the volume of the reaction solution to the reaction solution and allowing it to stand at 4℃ for 2h to precipitate; centrifuging at 8000rpm for 10min; washing the precipitate twice with a mixed solution of anhydrous ethanol and acetone (volume ratio 1.5:1); and vacuum drying at 60℃ for 6h to obtain zinc catechin complex.

[0058] The preparation method of modified chitosan-PEG gel includes: preparing a 3wt% chitosan solution with 2wt% acetic acid solution; adding dialdehyde polyethylene glycol at a mass ratio of chitosan:dialdehyde polyethylene glycol = 1:0.8; adjusting the pH to 6.5 with 0.8M NaOH aqueous solution; stirring at 50℃ and 200rpm for 4h to obtain a reaction solution; adding 1% sodium cyanoborohydride as a reducing agent and continuing the reaction for 1h under the same conditions to stabilize the Schiff base; then adding 3 times the volume of anhydrous ethanol to the reaction solution; allowing the solution to stand to precipitate; filtering; washing the precipitate twice with anhydrous ethanol; and vacuum drying at 45℃ for 6h to obtain modified chitosan (with no sodium cyanoborohydride residue); preparing a 5wt% modified chitosan solution with deionized water; adding glycerol to a final concentration of 0.5wt%; stirring at 30℃ and 100rpm for 3h; and adjusting the viscosity to 250Pa·s to obtain modified chitosan-PEG gel.

[0059] The preparation method of the above-mentioned multifunctional pharmaceutical composition for comprehensive dental restoration includes the following steps:

[0060] Keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, hydroxyapatite, and modified chitosan-PEG gel were mixed according to the specified mass ratios. 20 parts of deionized water were added, and the mixture was stirred evenly at 20°C. The pH was adjusted to 6.5 to form a gel, thus obtaining the pharmaceutical composition.

[0061] Comparative Example 1

[0062] The difference from Example 1 is that the keratin peptide was changed to 9.5 parts and the coral fungus extract was changed to 1 part.

[0063] Comparative Example 2

[0064] The difference from Example 1 is that the keratin peptide is a component with a value of less than 3 kDa.

[0065] Comparative Example 3

[0066] The difference from Example 1 is that in the preparation of the coral fungus extract, the subtilisin was replaced with serratiptase.

[0067] Comparative Example 4

[0068] The difference from Example 1 is that in the preparation of the coral fungus extract, bromelain is replaced with papain.

[0069] Comparative Example 5

[0070] The difference from Example 1 is that no bromelain is added in the preparation of the coral fungus extract.

[0071] Comparative Example 6

[0072] The difference from Example 1 is that no Bacillus subtilis protease is added in the preparation of the coral fungus extract.

[0073] Comparative Example 7

[0074] The difference from Example 1 is that in the preparation of the coral fungus extract, G-25 dextran gel was replaced with LH-20 dextran gel.

[0075] Comparative Example 8

[0076] The difference from Example 1 is that in the preparation of the coral extract, the ammonium acetate (NH4OAc) buffer was replaced with an 80 vol% aqueous ethanol solution as the eluent.

[0077] Comparative Example 9

[0078] The difference from Example 1 is that eggshell-derived nano-calcium phosphate is not added, and the amount of eggshell-derived nano-calcium phosphate is replaced by hydroxyapatite.

[0079] Comparative Example 10

[0080] The difference from Example 1 is that: the zinc catechin complex is not prepared, and the zinc catechin complex is directly replaced by catechin.

[0081] Comparative Example 11

[0082] The difference from Example 1 is that the step of "adding 0.7% sodium cyanoborohydride by mass of the reaction solution as a reducing agent and continuing the reaction for 1.5 h under the same conditions to stabilize the Schiff base" is omitted in the preparation of the modified chitosan-PEG gel.

[0083] The raw materials used in the above embodiments and comparative examples are as follows: Hydroxyapatite is from Zhengzhou Rongyuan Chemical Products Co., Ltd., nano-grade. Keratinase is from Shanxi Lanyuan Biotechnology Co., Ltd., with an enzyme activity of 200,000 U / g. Coral fungus is an edible *Cladosporium* species. Cellulase is from Jiangsu Changjing Bioengineering Co., Ltd., with an enzyme activity of 200,000 U / g. Bacillus subtilis protease is from Hefei Shengrun Biological Products Co., Ltd., with an enzyme activity of 50,000 U / g. Bromelain is from Shaanxi Zhenghe Pharmaceutical Bioengineering Co., Ltd., with an enzyme activity of 100,000 U / g. Catechins are from Hebei Baiwei Biotechnology Co., Ltd., with a purity of 99%. Chitosan (degree of deacetylation 95%) is from Xi'an Shouhe Biotechnology Co., Ltd., model SH-85-69. Dialdehyde polyethylene glycol is from Guangzhou Carbon Technology Co., Ltd., with a molecular weight of 2 kDa. Glycerol is from Xi'an Taihua Pharmaceutical Technology Co., Ltd., pharmaceutical grade glycerol. G-25 dextran gel and LH-20 dextran gel are from Shanghai Yuanye Biotechnology Co., Ltd. Serratiocin was derived from Leshengyuan Biotechnology (Nanjing) Co., Ltd., with an enzyme activity of 50,000 U / g. Papain was derived from Xi'an Lvteng Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0084] The above-mentioned drugs were subjected to the following tests.

[0085] The active pharmaceutical ingredient is defined as: a mixture of keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, and zinc catechin complex in a mass ratio; it does not contain modified chitosan-PEG gel and hydroxyapatite in the formulation; wherein, for comparative example 9, the eggshell-derived nano-calcium phosphate is replaced with hydroxyapatite, i.e., it contains 10 parts of hydroxyapatite.

[0086] The active ingredients of the drug are defined as: keratin peptides, coral fungus extract, eggshell-derived nano-calcium phosphate, zinc catechin complex, and hydroxyapatite mixed in a certain mass ratio; it does not contain modified chitosan-PEG gel.

[0087] I. HGF cytotoxicity detection:

[0088] Sample preparation: Take 0.1g of the active ingredient of the drug and add it to 10mL of DMEM complete medium containing 10% fetal bovine serum. Extract for 24h at 37℃ and 5% CO2. Filter through a 0.22μm filter membrane to obtain the stock solution. Dilute the stock solution with complete medium to a concentration of 50% (i.e., the volume ratio of stock solution to complete medium is 1:1) to obtain the working sample.

[0089] The detection method includes: human gingival fibroblasts (HGF) at 5 × 10⁻⁶ 3Cells were seeded per well in 96-well plates and cultured at 37°C and 5% CO2 for 24 h. The original culture medium was discarded, and the cells were washed once with PBS. 100 μL of the working sample was added to each well, with 5 replicates per group. A blank control group (containing only 100 μL of complete culture medium) and a negative control group (containing cells + 100 μL of complete culture medium) were included. The cells were cultured for another 24 h, and 10 μL of CCK-8 reagent was added to each well. The cells were incubated at 37°C for 2 h. The absorbance (OD) value at 450 nm was measured using a microplate reader. HGF cell viability (%) = (OD value of the working sample group - OD value of the blank control group) / (OD value of the negative control group - OD value of the blank control group) × 100%.

[0090] II. Antibacterial performance testing:

[0091] Strains: Streptococcus mutans (ATCC 25175), Porphyromonas gingivalis (ATCC 33277); prepared with PBS at a concentration of 1×10⁻⁶. 6 CFU / mL bacterial suspension.

[0092] Sample preparation: The active pharmaceutical ingredient was prepared into a working sample with a concentration of 2 mg / mL using PBS.

[0093] The detection method included setting up the following groups in a 96-well plate, with 5 replicates per group: Experimental group: 100 μL BHI broth medium + 100 μL working sample + 100 μL bacterial suspension per well; Positive control group: 100 μL BHI broth medium + 100 μL PBS + 100 μL bacterial suspension per well; Drug control group: 100 μL BHI broth medium + 100 μL working sample + 100 μL PBS (sterile) per well; Blank control group: 100 μL BHI broth medium + 200 μL PBS (drug-free and sterile) per well. Anaerobic incubation was performed at 37℃ for 24 h (80% N2, 10% H2, 10% CO2); OD values ​​at 600 nm were measured using a microplate reader. The inhibition rate was calculated using the formula: Inhibition rate (%) = [(OD positive control group - OD experimental group) / (OD positive control group - OD blank control group)] × 100%. The OD value of the experimental group is the value after deducting the OD value of the drug control group.

[0094] III. Mineralization performance testing:

[0095] Sample preparation: Take 0.2g of the active ingredient of the drug and add 10mL of simulated saliva (simulated saliva formula: CaCl2 1.5mmol / L, KH2PO4 0.9mmol / L, KCl 5.4mmol / L, NaN3 0.02% (w / v), pH 7.0), and stir magnetically for 30min until uniformly dispersed to obtain the working sample solution.

[0096] The testing method included: A 4mm × 4mm × 2mm ox tooth enamel block was polished and then demineralized (0.1 mol / L lactate buffer, pH 4.5, incubated at 37℃ for 72 h). Initial hardness was measured using a microhardness tester. Three test points were selected for each sample (200g load, 15s hold), and the average value was taken. Three parallel samples were prepared for each type. The enamel block was completely immersed in the working sample solution and incubated at 37℃ for 7 days, with the working sample solution replaced every 24 h. After removal, it was rinsed three times with deionized water, blotted dry with filter paper, and the hardness was measured using a microhardness tester. The hardness growth rate was calculated as: (mean HV after incubation - mean initial HV) / mean initial HV × 100%.

[0097] IV. Dentin tubule occlusion rate detection:

[0098] Sample: Gel-like pharmaceutical compositions (containing all ingredients) of Example 1 and Comparative Example 11.

[0099] The detection method included: removing enamel from bovine teeth to prepare dentin sheets (0.8cm × 0.8cm, 1mm thick), ultrasonically cleaning for 10 min; treating with 6% citric acid solution at room temperature for 2 min, rinsing three times with deionized water; then treating with 0.5mol / L EDTA solution at room temperature for 5 min to open the dentinal tubules, rinsing three times with deionized water, and drying with filter paper. A gel-like drug composition was uniformly coated onto the surface of the dentin sheet (0.5mm thick) and incubated at 37℃ and 100% relative humidity for 24 h. After incubation, deionized water at 37℃ was used to rinse the sample from 1cm away for 5 seconds at a constant flow rate of 5mL / s. Excess moisture was then gently blotted from the side of the sample with filter paper. SEM observation was performed, and ImageJ software was used to measure the total area of ​​dentinal tubule openings within the field of view (A0 before treatment, A1 after incubation). The dentinal tubule occlusion rate (%) was calculated as [(A0-A1) / A0] × 100%. Three parallel samples were used in each group, and the average value was taken.

[0100] Table 1. Test Results (Average Values)

[0101]

[0102] Note: "-" indicates that the corresponding indicator was not tested in this group.

[0103] The raw material ratios in Examples 1 to 3 are scientific, and the preparation of key components is compliant with regulations, achieving synergistic optimization of antibacterial, mineralization, repair, and dosage form stability. Keratin peptides and coral fungus extract supplement active peptides and polysaccharides to enhance antibacterial and mineralization effects. Eggshell-derived nano-calcium phosphate and hydroxyapatite synergistically and efficiently release mineralizing ions. Catechin zinc complex stabilizes antibacterial activity through coordination bonds, and modified chitosan-PEG gel ensures adhesion and sustained release, without functional imbalance caused by excessive or insufficient components. Keratin peptides, as mineralization templates, have abundant polar groups on their molecular chains, including carboxyl and hydroxyl groups, which can effectively capture calcium and phosphate ions, providing a large number of precise nucleation sites for the deposition of inorganic minerals, guiding orderly crystal growth, and forming a dense repair layer. Coral fungus extract, as a biomineralization inducer, contains polysaccharides, glycoproteins, and other components that can simulate the organic matrix of natural teeth. It can not only bind mineral ions but also regulate the local microenvironment, promote the transformation of amorphous minerals into crystalline states, and enhance the migration and enrichment of ions in the defect area. Keratin peptides are controlled to a range of 3kDa-10kDa, balancing permeability and stability; coral fungus extract is synergistically hydrolyzed by cellulase, subtilisin, and bromelain, and purified using G-25 dextran gel and ammonium acetate buffer to fully retain active peptides; eggshell-derived nano-calcium phosphate is precisely controlled to ensure mineralization efficiency; zinc catechin complex forms a stable structure; modified chitosan-PEG gel is stabilized with sodium cyanoborohydride to prevent gel disintegration; all processes are compatible, and the active ingredients are intact.

[0104] Comparative Example 1 (9.5 parts keratin peptide, 1 part coral fungus extract): Keratin peptide and coral fungus extract exhibit synergistic effects, enhancing antibacterial activity and aiding in mineralization. When keratin peptide is excessive, high concentrations of peptides increase the metabolic burden on cells, disrupting cell membrane stability and leading to decreased biocompatibility; insufficient coral fungus extract directly reduces the total amount of antibacterial and repairing active ingredients. Under these dual effects, cell survival rate and antibacterial rate are lower than in the previous example. The organic-inorganic composite mineralization system of keratin peptide and coral fungus extract is disrupted, reducing the speed and quality of remineralization, thereby decreasing the microhardness of the restored enamel.

[0105] Comparative Example 2 (keratin peptides below 3kDa): The keratin peptides required for tooth restoration need to balance permeability and stability: peptides between 3kDa and 10kDa can maintain structural stability, promote mineralization, and achieve long-lasting effects. Although small molecule peptides below 3kDa have increased permeability, they are easily degraded rapidly, making it difficult to form stable bonds with mineralized ions. At the same time, their destructive effect on bacterial cell membranes is weakened, ultimately leading to a decline in all performance indicators.

[0106] Comparative Examples 3 to 6 (Protein Replacement or Deletion in the Preparation of Coral Fungus Extract): The activity of coral fungus extract depends on the specific hydrolysis of proteins by proteases. Different enzymes have different cleavage sites, and variations in their enzymatic coordination strategies lead to different peptide chain structures, resulting in different efficacy and degree of effect. Protein replacement or deletion disrupts the types and amounts of active ingredients with optimal efficacy, leading to changes in various properties.

[0107] Comparative Example 7 (G-25 dextran gel replaced with LH-20 dextran gel in the preparation of coral fungus extract): The classification range and component types of LH-20 dextran gel and G-25 dextran gel differ, resulting in differences in the composition of the purified product. After replacing with LH-20 dextran gel, some small molecule active peptides were lost, and the residue of harmful impurities increased, ultimately resulting in lower performance indicators than in the example.

[0108] Comparative Example 8 (the eluent in the preparation of coral fungus extract was replaced with 80 vol% ethanol aqueous solution): Ammonium acetate buffer is a neutral water-soluble eluent that can maintain the spatial structure stability of active peptides, avoid denaturation, and specifically elute water-soluble active peptides; while 80 vol% ethanol aqueous solution is lipophilic, which will destroy the hydrophilic structure of active peptides. At the same time, it has poor elution specificity, and harmful impurities are eluted together with active peptides, ultimately resulting in a double decrease in the content and activity of active ingredients, affecting the efficacy.

[0109] Comparative Example 9 (without eggshell-derived nano-calcium phosphate, replaced by hydroxyapatite): Eggshell-derived nano-calcium phosphate is the core mineralization enhancing component: its nano-sized particles (D50 of 50-150nm) have a high specific surface area and can rapidly release Ca. 2+ With PO4 3- It synergistically promotes enamel remineralization with hydroxyapatite; however, hydroxyapatite has a slow ion release rate and lacks the surface activity of nanoparticles, making it difficult to form a stable bond with the enamel surface. It can only maintain the basic mineralization function, so the hardness growth rate is significantly lower than that of the example. Other indicators are similar to those of the example because the antibacterial and repair components remain unchanged.

[0110] Comparative Example 10 (no catechin zinc complex was prepared, and catechin was used directly instead): The catechin zinc complex achieves functional synergy through coordination bonds: zinc ions can enhance the anti-degradation ability of catechin, and at the same time, zinc ions themselves can destroy bacterial cell membranes and supplement antibacterial effects; while pure catechin is easily oxidized and inactivated in a humid environment, and does not have the synergistic antibacterial effect of zinc ions. Therefore, the inhibition rate against the two pathogenic bacteria is significantly lower than that in the example. The other indicators decreased less because the mineralization and repair components remained unchanged.

[0111] Comparative Example 11 (The sodium cyanoborohydride reduction step was omitted in the preparation of the modified chitosan-PEG gel):

[0112] The core function of sodium cyanoborohydride is to reduce the Schiff base formed by chitosan and dialdehyde polyethylene glycol, converting it into a stable CN bond, thereby enhancing the structural stability and bioadhesion of the gel and ensuring that the gel does not disintegrate or fall off in a humid environment. Without this, the Schiff base is easily hydrolyzed, the gel is easily detached during the rinsing step in the test, and the drug release is too rapid, which cannot effectively block the dentinal tubules. Therefore, the dentinal tubule blocking rate is significantly lower than that in Example 1.

Claims

1. A multifunctional pharmaceutical composition for comprehensive dental restoration, characterized in that, It is composed of the following raw materials in mass fraction: keratin peptide 5-8 parts, Ramaria botrytis (Pers.) Ricken extract 3-5 parts, eggshell-derived nano calcium phosphate 8-12 parts, catechin zinc complex 2-3 parts, hydroxyapatite 10-15 parts, modified chitosan-PEG gel 60-80 parts and deionized water, pH 6.5-7.5; The preparation method of the keratin peptide comprises the following steps: soaking white goose down in NaOH aqueous solution, filtering, washing, drying, crushing into powder, adding 8-12 times the mass of the powder in deionized water, adjusting pH to 8.0-9.0, adding 2-3% of the mass of the powder of keratinase, carrying out enzymolysis at 50-55℃ for 2.5-3.5 hours, inactivating the enzyme, centrifuging, taking the supernatant, ultrafiltrating with an ultrafiltration membrane, taking the component between 3kDa and 10kDa, and freeze-drying to obtain the keratin peptide; The preparation method of the Ramaria botrytis (Pers.) Ricken extract comprises the following steps: crushing Ramaria botrytis (Pers.) Ricken into fungus powder, adding 8-12 times the mass of the fungus powder in deionized water, adjusting pH to 4.5-5.5, adding 1-1.5% of the mass of the fungus powder of cellulase, carrying out enzymolysis at 50-55℃ for 1.5-2 hours, adjusting pH to 6.5-7.5, adding 1-1.5% of the mass of the fungus powder of subtilisin and 1-1.5% of the mass of the fungus powder of bromelain, carrying out enzymolysis at 50-55℃ for 2-2.5 hours, inactivating the enzyme, centrifuging, taking the supernatant, concentrating under reduced pressure, loading onto a G-25 dextran gel column, washing with deionized water to remove impurities, eluting with ammonium acetate buffer, collecting the eluate, concentrating under reduced pressure, and freeze-drying to obtain the Ramaria botrytis (Pers.) Ricken extract; The eggshell-derived nano calcium phosphate is obtained by the following steps: dissolving eggshell powder in acetic acid aqueous solution to form calcium acetate solution, reacting with NaOH aqueous solution to form calcium hydroxide precipitate, and then reacting with disodium hydrogen phosphate to form eggshell-derived nano calcium phosphate; and the catechin zinc complex is obtained by the reaction of catechin and zinc sulfate; The preparation method of the modified chitosan-PEG gel comprises the following steps: preparing a chitosan solution with a concentration of 3-5 wt% by using 1-2 wt% acetic acid solution, adding dialdehyde polyethylene glycol according to the mass ratio of chitosan:dialdehyde polyethylene glycol=1:(0.5-0.8), adjusting pH to 6.0-6.5 with NaOH aqueous solution, and stirring and reacting at 50-70℃ and 100-200 rpm for 4-8 hours to obtain a reaction solution, adding sodium cyanoborohydride for further reaction, adding anhydrous ethanol, standing to precipitate, filtering, washing the precipitate with anhydrous ethanol, drying to obtain modified chitosan, preparing a modified chitosan solution with a concentration of 2-5 wt% by using deionized water, adding glycerol with a final concentration of 0.5-2 wt%, stirring, and adjusting the viscosity to 150-250 Pa·s to obtain the modified chitosan-PEG gel.

2. The multifunctional pharmaceutical composition for the comprehensive repair of teeth according to claim 1, characterized in that, In the preparation method of the keratin peptide, the concentration of the aqueous NaOH solution is 0.5-1.0 mol / L; the soaking is carried out at 50-70 DEG C for 2-3 hours; the washing is carried out with deionized water until the pH is 6.5-7.0; the enzyme inactivation is carried out at 90-95 DEG C for 10-15 minutes; and the centrifugation is carried out at 6000-8000 rpm for 10-20 minutes.

3. The multifunctional pharmaceutical composition for the comprehensive tooth restoration according to claim 1, characterized in that, In the preparation method of the coral fungus extract, the coral fungus is dried coral fungus fruiting body; the particle size of the fungus powder is 80-100 mesh; the enzyme inactivation is carried out at 85-90 DEG C for 10-15 minutes and then the temperature is decreased to room temperature; and the centrifugation is carried out at 6000-8000 rpm for 10-20 minutes.

4. The multifunctional pharmaceutical composition for the comprehensive tooth repair according to claim 1, characterized in that, The preparation method of the eggshell-derived nano calcium phosphate comprises the following steps: crushing goose eggshells into eggshell powder, adding the eggshell powder into 1-2 M aqueous acetic acid solution, stirring, removing insoluble substances by filtration, obtaining calcium acetate solution, adjusting the pH to 11.0-12.0 by using 1-2 M aqueous NaOH solution, generating calcium hydroxide precipitate, washing with deionized water, resuspending with deionized water, obtaining calcium hydroxide suspension, adding 0.1-1 M aqueous disodium hydrogen phosphate solution, controlling the calcium-phosphorus molar ratio to be (1.5-1.7):1, adjusting the pH to 9.0-10.5 by using 0.1-1 M aqueous NaOH solution, stirring and reacting, centrifuging, washing the precipitate with deionized water until the pH is 6.5-7.5, drying, and dry ball-milling until the particle size D50 is 50-150 nm, to obtain eggshell-derived nano calcium phosphate.

5. The multifunctional pharmaceutical composition for the comprehensive tooth repair according to claim 1, characterized in that, The preparation method of the catechin zinc complex comprises the following steps: preparing 5-8 wt% catechin solution by using deionized water, preparing 0.1-0.2 M zinc sulfate solution by using deionized water, adding the zinc sulfate solution into the catechin solution according to the mass ratio of catechin to zinc in zinc sulfate being 1:(0.18-0.25), adjusting the pH to 7.0-7.5, stirring and reacting in the dark, adding anhydrous ethanol, standing and precipitating, centrifuging, washing the precipitate with a mixed solution of anhydrous ethanol and acetone, and vacuum drying, to obtain catechin zinc complex.

6. The multifunctional pharmaceutical composition for the comprehensive tooth repair according to claim 1, wherein In the preparation method of the modified chitosan-PEG gel, the aqueous NaOH solution is 0.8-1 M aqueous NaOH solution; and the amount of the anhydrous ethanol is 2-3 times the volume of the reaction solution.

7. The method of preparing a multi-functional dental restorative pharmaceutical composition according to claim 1, wherein, The method comprises the following steps: The drug composition is obtained by mixing the keratin peptide, the coral fungus extract, the eggshell-derived nano calcium phosphate, the catechin zinc complex, the hydroxyapatite and the modified chitosan-PEG gel according to the mass fraction, adding 10-20 parts of deionized water, stirring uniformly at 20-35 DEG C, adjusting the pH to 6.5-7.5, and forming a gel.

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