Active pharmaceutical ingredient for the repair of oral mucosa and method for its preparation

Abstract

The present application relates to a kind of active pharmaceutical ingredients of repairing oral mucosa and preparation method thereof, belong to biological medicine technical field, and drug includes orange cover gonggao mycelium active peptide- Terminalia polyphenol composite package, dendrobium nobile callus extract, sorbitol, menthol, gel matrix etc.Orange cover gonggao mycelium active peptide- Terminalia polyphenol composite package is the complex of orange cover gonggao mycelium active peptide and Terminalia polyphenol by sodium alginate-dopamine graft copolymer package;Orange cover gonggao mycelium active peptide is the mycelium of orange cover gonggao mushroom by bromelain and bacillus subtilis protease common enzymolysis obtains.Golden lace dendrobium callus extract is the section of golden lace dendrobium seedling leaf by 2,4-D and 6-BA induction, extraction, purification and obtains.Gel matrix is prepared by modified konjac glucomannan and carboxymethyl chitosan.Each component forms anti-inflammatory, proliferation and remodeling closed-loop repair system, and improves the stability of anti-degradation and adheres long-acting.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an active pharmaceutical ingredient for repairing oral mucosa and its preparation method. Background Technology

[0002] The oral mucosa is a soft tissue barrier covering the inner wall of the mouth, and its integrity is crucial for oral health. Because the oral mucosa is directly exposed to mechanical friction, chemical irritation, microbial invasion, and physical damage, it is highly susceptible to damage, leading to oral ulcers, erosions, or inflammation.

[0003] Oral mucosal repair is a multi-stage, synergistic process: First, the inflammatory phase occurs, during which blood vessels dilate and immune cells gather at the site of damage, clearing necrotic tissue and inhibiting infection. Second, the proliferative phase occurs, during which mucosal epithelial cells rapidly divide and migrate, covering the wound, while fibroblasts synthesize collagen to build a repair scaffold. Finally, the remodeling phase occurs, during which newly formed tissue gradually differentiates and matures, restoring the structure and function of the mucosa. Under normal circumstances, healthy oral mucosa, due to its rich blood circulation and rapid epithelial cell renewal, can heal minor injuries spontaneously in about 7-14 days. However, when the area of ​​mucosal damage is large, the patient's own repair ability is reduced, or there are persistent irritants, the repair process can be hindered, leading to chronic mucosal lesions.

[0004] Medications for repairing oral mucosa have undergone a functional upgrade from symptomatic relief to promoting repair. Early intervention focuses on symptom relief, such as using local anesthetics containing lidocaine or benzocaine to reduce pain, or antibacterial drugs containing chlorhexidine or cetirizine to prevent infection. However, these drugs only temporarily improve discomfort and cannot accelerate mucosal repair, with limited effectiveness for chronic or large-area damage. With a deeper understanding of the mechanisms of mucosal repair, drugs have gradually developed towards actively promoting healing: one type is drugs that supplement the nutrients needed for repair, such as preparations containing vitamin B2, vitamin C, and zinc, which enhance the proliferation capacity of epithelial cells by improving mucosal cell metabolism; another type is drugs that mimic the mucosal protective barrier, such as gels or films containing chitosan, hyaluronic acid, and alginate, which can form a physical protective film on the wound, isolating irritants while providing a moist environment for mucosal repair. These drugs have become commonly used adjunctive repair drugs in clinical practice.

[0005] In recent years, with the development of cell biology and materials science, drugs for repairing oral mucosa have been further upgraded to target and regulate the repair process. For example, drugs containing growth factors can directly activate the proliferation signaling pathways of epithelial cells and fibroblasts, significantly shortening wound healing time. Novel formulations containing stem cell exosomes and bioactive peptides can address the problem of traditional drugs' poor efficacy on difficult-to-heal wounds by regulating inflammatory responses and promoting angiogenesis. However, existing drugs still have limitations: some drugs have poor stability and are easily degraded; some patch-type drugs have insufficient adhesion and are prone to falling off during chewing; at the same time, the repair efficiency of existing drugs still needs to be improved. These clinical needs are driving the continuous research and development of drugs for repairing oral mucosa towards greater stability and longer-lasting effects. Summary of the Invention

[0006] To address the shortcomings in existing technologies for repairing oral mucosa, such as insufficient efficacy, anti-degradation stability, and long-lasting adhesion, this invention provides an active pharmaceutical ingredient for repairing oral mucosa and its preparation method. The main active ingredients are an active peptide-Terminalia chebula polyphenol complex carrier and Dendrobium nobile callus extract, forming a closed-loop repair system promoting anti-inflammation, proliferation, and remodeling, thus enhancing the repair effect. Furthermore, the carrier improves anti-degradation stability, and a modified gel matrix is ​​designed to improve adhesion and long-lasting adhesion, continuously providing the effect of repairing the mucosa. The specific technical solution is as follows:

[0007] An active pharmaceutical ingredient for repairing oral mucosa, comprising the following raw materials in parts by weight: 8-10 parts of Amanita fulva mycelium active peptide-Terminalia chebula polyphenol complex carrier, 4-6 parts of Dendrobium nobile callus extract, 3-5 parts of sorbitol, 0.1-0.3 parts of menthol, 25-35 parts of gel matrix, 0.1-0.3 parts of citric acid, and the balance being deionized water, totaling 100 parts;

[0008] The active peptide-Terminalia chebula polyphenol composite loading material is obtained by loading the active peptide of Amanita tangutica mycelium and the polyphenol of Terminalia chebula onto a sodium alginate-dopamine graft copolymer and then cross-linking and curing it with calcium chloride; the mass ratio of the active peptide of Amanita tangutica mycelium to the polyphenol of Terminalia chebula is (6-7):(3-4).

[0009] The active peptides from the mycelium of Amanita fulva are products with a value between 1 kDa and 5 kDa obtained by enzymatic hydrolysis of the mycelium of Amanita fulva with bromelain and Bacillus subtilis protease; the polyphenols from Terminalia chebula are obtained by extraction of Terminalia chebula fruit with ethanol aqueous solution, and purification of the extract by AB-8 macroporous adsorption resin column; the sodium alginate-dopamine graft copolymer is obtained by reaction of sodium alginate with dopamine hydrochloride after activation by EDC and NHS.

[0010] The Dendrobium nobile callus extract was obtained by inducing Dendrobium nobile seedling leaf slices in MS liquid medium with 2,4-D and 6-BA, followed by dark culture and light culture. The callus tissue was then extracted with ethanol aqueous solution, and the extract was adsorbed through an AB-8 macroporous resin column. The eluent was then allowed to stand with acetone to obtain the precipitate.

[0011] The viscosity of the gel matrix is ​​5000 mPa·s to 8000 mPa·s; the gel matrix is ​​obtained by reacting modified konjac glucomannan, carboxymethyl chitosan, genipin and EDTA-2Na; the mass ratio of modified konjac glucomannan to carboxymethyl chitosan is (2-3):(1.4-1.8); the modified konjac glucomannan is obtained by hydroxypropylation modification of konjac glucomannan with propylene oxide.

[0012] The preparation method of the above-mentioned active peptide-Terminalia chebula polyphenol complex loading material from Amanita tangutica mycelium includes: mixing active peptides from Amanita tangutica mycelium and Terminalia chebula polyphenols at a mass ratio of (6-7):(3-4) to obtain a complex; taking sodium alginate-dopamine graft copolymer, and preparing an internal aqueous phase by mixing the complex and sodium alginate-dopamine graft copolymer together with deionized water at a mass ratio of complex:sodium alginate-dopamine graft copolymer = 1:(1.2-1.5); taking a mixture containing 1wt%-3wt% Liquid paraffin from Span-80 was used as the oil phase. The aqueous phase was added dropwise to the oil phase under high-speed homogenization at 8000 rpm–10000 rpm, and the mixture was continuously homogenized at high speed to form an emulsion. The emulsion was then added to a 50 vol%–60 vol% ethanol aqueous solution containing 2 wt%–5 wt% calcium chloride under continuous stirring at 400 rpm–600 rpm. The reaction proceeded to form solid microspheres. After standing, the precipitate was collected, washed alternately with anhydrous ethanol and ether, vacuum dried, and sieved to obtain an active peptide-Terminalia chebula polyphenol complex from Amanita fulva mycelium.

[0013] The preparation method of the above-mentioned active peptides from Amanita fulva mycelium includes: washing the mycelium of Amanita fulva with physiological saline, then diluting it with deionized water to form a mycelium suspension, and ultrasonically disrupting and homogenizing it; adjusting the pH to 6.5-7.2 and the temperature to 50℃-55℃, adding bromelain and Bacillus subtilis protease, enzymatically hydrolyzing for 2-3 hours, inactivating the enzymes, cooling, centrifuging, taking the supernatant, ultrafiltration with an ultrafiltration membrane to obtain a fraction between 1kDa and 5kDa, and freeze-drying to obtain active peptides from Amanita fulva mycelium.

[0014] In the above-mentioned method for preparing active peptides from Amanita fulva mycelium, the mycelium is obtained by culturing Amanita fulva in a liquid fermentation medium; the amount of bromelain and Bacillus subtilis protease added is 0.15% to 0.25% of the mass of the mycelium suspension; the enzyme inactivation is performed at 85℃ to 90℃ for 10 to 15 minutes; and the centrifugation is performed at 9000 rpm to 10000 rpm for 10 to 15 minutes.

[0015] The preparation method of the above-mentioned Terminalia chebula polyphenols includes: pulverizing dried Terminalia chebula fruit and extracting it by reflux with a 70 vol%–80 vol% ethanol aqueous solution at 70℃–80℃; concentrating the extract under reduced pressure and then transferring it to an AB-8 macroporous adsorption resin column; first rinsing with deionized water for 3 BV–5 BV to remove impurities, then eluting with a 60 vol%–70 vol% ethanol aqueous solution for 4 BV–5 BV; collecting the eluent, concentrating under reduced pressure, and drying under vacuum to obtain Terminalia chebula polyphenols.

[0016] The preparation method of the above-mentioned sodium alginate-dopamine graft copolymer includes: preparing a 15 g / L to 20 g / L sodium alginate solution using MES buffer solution with pH 5.5 to 6.5, adding 8 g / L to 12 g / L EDC and 5 g / L to 7 g / L NHS, activating the carboxyl groups under nitrogen protection and light protection at room temperature by stirring, and obtaining an activated solution; dissolving dopamine hydrochloride in 2 to 3 times its mass of DMSO to obtain a dopamine hydrochloride solution; adding the dopamine hydrochloride solution to the activated solution under nitrogen protection, light protection, and stirring at room temperature according to the mass ratio of sodium alginate:dopamine hydrochloride = 10:(2.5 to 3.5), reacting, and obtaining a reaction solution; adding the solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing with deionized water, filtering, and freeze-drying the filtrate to obtain the sodium alginate-dopamine graft copolymer.

[0017] The preparation method of the above-mentioned Dendrobium nobile callus extract includes: taking sterile leaves of Dendrobium nobile seedlings, cutting them into small pieces, placing them in MS liquid medium containing 0.5 mg / L to 1.5 mg / L of 2,4-D and 0.2 mg / L to 0.8 mg / L of 6-BA, and culturing them at 24℃ to 26℃ in the dark for 10 to 20 days to induce callus formation, replacing the medium with fresh medium, and culturing at 24℃ to 26℃ under light for 7 to 10 days; and then collecting the callus tissue. Extraction was performed by reflux with 70 vol%–80 vol% ethanol aqueous solution at 70℃–80℃, followed by vacuum concentration, filtration, and loading of the filtrate onto an AB-8 macroporous resin column. The column was first eluted with deionized water for 3 BV–5 BV to remove impurities, and then eluted with 80 vol%–90 vol% ethanol aqueous solution for 4 BV–5 BV. The eluent was collected, concentrated under reduced pressure to obtain an extract, and acetone was added for precipitation. The extract was then centrifuged, collected, and vacuum dried to obtain the Dendrobium nobile callus extract.

[0018] The preparation method of the above-mentioned gel matrix includes: mixing modified konjac glucomannan and carboxymethyl chitosan at a mass ratio of (2-3):(1.4-1.8), and preparing a solution with purified water; adding 8%-10% genipin by mass of modified konjac glucomannan, and stirring to react; adding 2%-3% EDTA-2Na by mass of modified konjac glucomannan, stirring, and adjusting the viscosity to 5000 mPa·s-8000 mPa·s to obtain the gel matrix.

[0019] The preparation method of the above-mentioned modified konjac glucomannan includes: adding konjac glucomannan to isopropanol, stirring and dispersing, adding 0.5% to 1% sodium hydroxide by weight of konjac glucomannan, adding 1 to 2 times the weight of propylene oxide by weight of konjac glucomannan, reacting, adding hydrochloric acid to terminate the reaction, filtering, washing the precipitate with ethanol, and vacuum drying to obtain modified konjac glucomannan.

[0020] The preparation method of the above-mentioned active pharmaceutical ingredient for repairing oral mucosa includes the following steps:

[0021] According to the mass fractions, add the active peptide-Terminalia chebula polyphenol complex carrier of Amanita tangutica mycelium, Dendrobium nobile callus extract, sorbitol, menthol, gel matrix, and citric acid to deionized water, stir and mix evenly, and then degas under vacuum to form a gel.

[0022] The above-mentioned gel is used as a medication for repairing oral mucosa.

[0023] This invention provides an active pharmaceutical ingredient for repairing oral mucosa and its preparation method, with the following beneficial effects:

[0024] I. Through multi-component synergy and precise process control, the medication optimizes the entire process of oral mucosal repair. It covers the inflammatory, proliferative, and remodeling phases of oral mucosal repair, overcoming the limitations of traditional medications that only relieve symptoms or repair a single stage, thus forming a closed-loop repair system.

[0025] Second, by encapsulating the active ingredients with sodium alginate-dopamine graft copolymer, the active peptides are prevented from being enzymatically degraded and the polyphenols from being oxidized, while slow release is achieved, ensuring the continuous action time of the drug in the oral cavity and solving the problems of poor stability and easy degradation of traditional growth factor drugs.

[0026] Third, the high-viscosity gel matrix, after hydroxypropylation modification, has excellent mucosal adhesion and mechanical stability. It can form a continuous adhesive layer under mechanical friction environments such as chewing, which isolates food, microorganisms and other irritants, while providing a moist environment for the wound, thus solving the defects of traditional films that are easy to fall off and do not provide long-lasting protection.

[0027] IV. Active Peptides from Amanita fulva Mycelium and Terminalia chebula Polyphenol Complex Encapsulation: Amanita fulva mycelium is enzymatically hydrolyzed using bromelain and Bacillus subtilis protease, utilizing the different cleavage sites of the two enzymes to obtain specific active peptides. These active peptides can promote wound coverage by activating epithelial cell proliferation signaling pathways, while Terminalia chebula polyphenols regulate inflammation by scavenging ROS and directly inhibiting inflammatory cells. The two form a synergistic effect of proliferation and protection. After encapsulation with sodium alginate-dopamine graft copolymer, the biocompatibility of sodium alginate and the adhesiveness of dopamine further enhance the cell affinity and stability of the active ingredients, reducing toxicity.

[0028] V. Dendrobium nobile callus extract: Through the induction of callus formation in Dendrobium nobile leaves by 2,4-D and 6-BA, polysaccharides, alkaloids and other repair active ingredients can be directionally accumulated. After extraction, the extract is purified by AB-8 macroporous resin column. The obtained components can directly activate fibroblasts, promote collagen synthesis, and assist in anti-inflammation. It complements the complex loading material in epithelial repair and fibroblast activation.

[0029] VI. Gel Matrix: After hydroxypropylation modification, the molecular chain flexibility of konjac glucomannan is improved. It is cross-linked with carboxymethyl chitosan through genipin and the viscosity is adjusted by EDTA-2Na to form a highly adhesive gel. This gel can fix the active ingredients on the wound surface and resist mechanical friction, providing a stable microenvironment for repair and preventing the active ingredients from being lost with saliva.

[0030] VII. Auxiliary ingredients such as sorbitol, menthol, and citric acid: Sorbitol acts as a moisturizer to maintain the moisture of the gel; menthol relieves pain through a local cooling sensation; and citric acid adjusts the pH to be close to the physiological environment of the oral cavity.

[0031] In summary, the synergistic effect of this drug formulation stems from the complementary functions of its components and the compatibility between the process and the ingredients. The active peptides from *Amanita fulva* mycelium, *Terminalia chebula* polyphenols, and *Dendrobium nobile* callus extract form a synergistic closed loop of epithelial repair, inflammation regulation, and tissue remodeling, working together to cover the entire repair process. The sodium alginate-dopamine graft copolymer, after encapsulating the active ingredients, not only protects the active peptides and polyphenols from degradation and enhances stability, but also strengthens the binding between the encapsulated material and the gel matrix through the adhesiveness of dopamine. The slow-release characteristics of the microspheres maintain the concentration of the active ingredients within an effective and non-toxic range, which, combined with the local retention effect of the gel matrix, achieves a synergistic effect of continuous release and high local concentration, improving bioavailability. The high adhesiveness and wettability of the modified gel matrix not only fix the active ingredients on the wound surface, preventing them from being lost with saliva, but also promote epithelial cell migration through a moist environment; simultaneously, the physical barrier effect of the gel matrix isolates external stimuli, reducing secondary damage to the wound surface, forming a synergistic protective and repairing effect with the repairing action of the active ingredients, accelerating healing. Detailed Implementation

[0032] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0033] Definitions:

[0034] MES buffer: 2-morpholinoethanesulfonic acid buffer;

[0035] EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0036] NHS: N-hydroxysuccinimide;

[0037] DMSO: Dimethyl sulfoxide:

[0038] 2,4-D: 2,4-Dichlorophenoxyacetic acid;

[0039] 6-BA: 6-Benzylaminopurine;

[0040] EDTA-2Na: Disodium ethylenediaminetetraacetate.

[0041] Example 1

[0042] An active pharmaceutical ingredient for repairing oral mucosa, comprising the following raw materials in parts by weight: 9 parts of active peptide-Terminalia chebula polyphenol complex carrier, 5 parts of Dendrobium nobile callus extract, 4 parts of sorbitol, 0.2 parts of menthol, 30 parts of gel matrix, 0.2 parts of citric acid, and the balance being deionized water, totaling 100 parts.

[0043] The preparation method of active peptides from Amanita fulva mycelium includes: inoculating Amanita fulva into a liquid fermentation medium (formula: glucose 30 g / L, soybean meal 15 g / L, KH2PO4 2 g / L, MgSO4·7H2O) under aseptic conditions. 1 g / L (with the remainder being water, pH at rest) was used for aerobic culture at 28℃ and 150 rpm for 6 days to obtain mycelial culture medium. The mycelium was collected by centrifugation at 9000 rpm for 15 min, washed twice with physiological saline, and then diluted with deionized water to a mycelial suspension equal to 7 times the mycelial mass. The suspension was then sonicated for 35 min under ultrasonic conditions of 400W power, 2 s working time, and 4 s intermittent time. The pH was adjusted to 6.8 and the temperature to 52℃. Bromelain and Bacillus subtilis protease, each at 0.2% by weight, were added to the mycelial suspension for enzymatic hydrolysis for 2.5 h. The enzymes were inactivated at 88℃ for 12 min, cooled, and centrifuged at 9500 rpm for 12 min. The supernatant was collected and ultrafiltered using an ultrafiltration membrane to obtain fractions between 1 kDa and 5 kDa. The fractions were then freeze-dried to obtain active peptides from Amanita fulva mycelium.

[0044] The preparation method of Terminalia chebula polyphenols includes: taking dried Terminalia chebula fruit, crushing it through a 40-mesh sieve, and extracting it twice at 75℃ for 1.5 hours each time with a 75 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:12. The extracts are combined, concentrated under reduced pressure to remove ethanol and some water, and then poured onto an AB-8 macroporous adsorption resin column. The column is first washed with deionized water for 4 BV to remove impurities, and then eluted with 65 vol% ethanol aqueous solution for 4.5 BV. The eluent is collected, concentrated under reduced pressure at 55℃, and then vacuum dried to obtain Terminalia chebula polyphenols.

[0045] The preparation method of sodium alginate-dopamine graft copolymer includes: preparing an 18 g / L sodium alginate solution using MES buffer at pH 6.0, adding 10 g / L EDC and 6 g / L NHS, and activating the carboxyl groups by stirring under nitrogen protection and in the dark at room temperature for 45 min to obtain an activated solution; dissolving dopamine hydrochloride in 2.5 times its mass of DMSO to obtain a dopamine hydrochloride solution; adding the dopamine hydrochloride solution to the activated solution at a mass ratio of sodium alginate:dopamine hydrochloride = 10:3 under nitrogen protection, in the dark, and at room temperature by stirring, and reacting for 20 h to obtain a reaction solution; adding the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing with deionized water for 60 h, filtering through a 0.22 μm microporous membrane, and freeze-drying the filtrate to obtain the sodium alginate-dopamine graft copolymer.

[0046] The preparation method of the active peptide-Terminalia chebula polyphenol complex loading material from Amanita tangutica mycelium includes: mixing active peptides from Amanita tangutica mycelium and Terminalia chebula polyphenols at a mass ratio of 6.5:3.5 to obtain a complex; preparing an internal aqueous phase by mixing the complex and the sodium alginate-dopamine graft copolymer together with deionized water at a mass ratio of total mass:water = 1:3.5 according to a mass ratio of complex:sodium alginate-dopamine graft copolymer = 1:1.3; and using liquid paraffin containing 2wt% Span-80 as the oil phase. Under high-speed homogenization at 9000 rpm, the aqueous phase was added dropwise to the oil phase at a volume ratio of 1:5, and homogenization was continued at high speed for 4 min to form an emulsion. Under continuous stirring at 500 rpm, the emulsion was added to 2.5 times its volume of a 55 vol% ethanol aqueous solution containing 3 wt% calcium chloride, and the reaction was carried out for 30 min to form solid microspheres. After standing, the precipitate was collected and washed twice alternately with anhydrous ethanol and diethyl ether. After vacuum drying at 42℃ for 6 h, the mixture was passed through a 100-mesh sieve to obtain the active peptide-Terminalia chebula polyphenol complex loaded with Amanita tangutica mycelium.

[0047] The preparation method of Dendrobium nobile callus extract includes: taking sterile leaves of Dendrobium nobile seedlings, cutting them into small pieces, and placing them in MS liquid medium containing 1 mg / L 2,4-D and 0.5 mg / L 6-BA. Culture in the dark at 25℃ for 15 days to induce callus formation. Discard the old medium and add new medium (same as above), and culture in the light at 25℃ for 8 days. Take the callus and extract it twice with 75 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:12 at 75℃, refluxing each time. After 5 hours, the extracts were combined, concentrated under reduced pressure to remove ethanol and some water, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto an AB-8 macroporous resin column. The column was first washed with deionized water for 4 BV to remove impurities, and then eluted with 85 vol% ethanol aqueous solution for 4.5 BV. The eluent was collected and concentrated under reduced pressure at 55 °C to obtain an extract. Three times the mass of the extract in acetone was added, and the column was allowed to stand at 5 °C for 14 hours to precipitate. The column was then centrifuged at 4000 r / min for 15 min, the precipitate was collected, and the precipitate was dried under vacuum at 50 °C to obtain the Dendrobium nobile callus extract.

[0048] The preparation method of the gel matrix includes: taking konjac glucomannan, adding isopropanol at a mass ratio of 1:9, stirring and dispersing, adding 0.8% sodium hydroxide (catalyst) of konjac glucomannan by mass, adding 1.5 times the mass of konjac glucomannan by mass of propylene oxide, reacting at 40℃ under nitrogen protection for 3h (hydroxypropylation modification), adding hydrochloric acid to a final concentration of 0.5mol / L to terminate the reaction, filtering, washing the precipitate twice with ethanol, and vacuum drying at 60℃ for 6h to obtain modified konjac glucomannan; mixing modified konjac glucomannan and carboxymethyl chitosan at a mass ratio of 2.5:1.6, and preparing a solution with purified water at a mass ratio of 1:7; adding 9% genipin of modified konjac glucomannan by mass, stirring and reacting at 45℃ for 25min; adding 2.5% EDTA-2Na of modified konjac glucomannan by mass, stirring for 8min, adjusting the viscosity to 6500mPa·s to obtain the gel matrix.

[0049] The preparation method of the above-mentioned active pharmaceutical ingredient for repairing oral mucosa includes the following steps:

[0050] The active peptides of Amanita fulva mycelium-Terminalia chebula polyphenol complex, Dendrobium nobile callus extract, sorbitol, menthol, gel matrix, and citric acid were added to deionized water, stirred and mixed evenly, and then vacuum degassed to form a gel.

[0051] Example 2

[0052] An active pharmaceutical ingredient for repairing oral mucosa, comprising the following raw materials in parts by weight: 10 parts of active peptide-Terminalia chebula polyphenol complex carrier, 4 parts of Dendrobium nobile callus extract, 5 parts of sorbitol, 0.1 parts of menthol, 35 parts of gel matrix, 0.1 parts of citric acid, and the balance being deionized water, totaling 100 parts.

[0053] The preparation method of active peptides from Amanita fulva mycelium includes: inoculating Amanita fulva into a liquid fermentation medium (formula: glucose 35 g / L, soybean meal 10 g / L, KH2PO4 3 g / L, MgSO4·7H2O) under aseptic conditions. 0.5 g / L (with the remainder being water, pH at natural), and aerobic culture was carried out at 30℃ and 120 rpm for 7 days to obtain mycelial culture medium; the mycelium was collected by centrifugation at 8000 rpm for 20 min, washed twice with physiological saline, and then diluted with deionized water to a mycelial suspension equal to 8 times the mycelial mass. The mycelial suspension was then ultrasonically homogenized for 40 min at 300 W power, with a working time of 3 seconds and an intermittent time of 3 seconds; the pH was adjusted to 6.5 and the temperature to 55℃, and 0.15% of bromelain and Bacillus subtilis protease were added to the mycelial suspension for 3 h of enzymatic hydrolysis; the enzymes were inactivated at 85℃ for 15 min, cooled, and centrifuged at 9000 rpm for 15 min to collect the supernatant. The supernatant was ultrafiltered using an ultrafiltration membrane to obtain a fraction between 1 kDa and 5 kDa, which was then freeze-dried to obtain active peptides from Amanita fulva mycelium.

[0054] The preparation method of Terminalia chebula polyphenols includes: taking dried Terminalia chebula fruit, crushing it through a 60-mesh sieve, and extracting it three times at 70℃ with a 70 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:15 for 1 hour each time. The extracts are combined, concentrated under reduced pressure to remove ethanol and some water, and then poured onto an AB-8 macroporous adsorption resin column. The column is first washed with deionized water to remove 5 BV of impurities, and then eluted with 60 vol% ethanol aqueous solution to remove 5 BV. The eluent is collected, concentrated under reduced pressure at 50℃, and dried under vacuum to obtain Terminalia chebula polyphenols.

[0055] The preparation method of sodium alginate-dopamine graft copolymer includes: preparing a 15 g / L sodium alginate solution with pH 6.5 MES buffer, adding 12 g / L EDC and 5 g / L NHS, and activating the carboxyl groups by stirring under nitrogen protection and in the dark at room temperature for 60 min to obtain an activated solution; dissolving dopamine hydrochloride in 2 times its mass of DMSO to obtain a dopamine hydrochloride solution; adding the dopamine hydrochloride solution to the activated solution at a mass ratio of sodium alginate:dopamine hydrochloride = 10:3.5 under nitrogen protection, in the dark, and stirring at room temperature, and reacting for 12 h to obtain a reaction solution; adding the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing with deionized water for 72 h, filtering through a 0.22 μm microporous membrane, and freeze-drying the filtrate to obtain the sodium alginate-dopamine graft copolymer.

[0056] The preparation method of the active peptide-Terminalia chebula polyphenol complex loading material from Amanita tangutica mycelium includes: mixing active peptides from Amanita tangutica mycelium and Terminalia chebula polyphenols at a mass ratio of 6:4 to obtain a complex; taking sodium alginate-dopamine graft copolymer, and mixing the complex and sodium alginate-dopamine graft copolymer together with deionized water at a mass ratio of total mass to water of 1:4 to prepare an internal aqueous phase; taking a 1wt%... Liquid paraffin from Span-80 was used as the oil phase. Under high-speed homogenization at 10,000 rpm, the aqueous phase was added dropwise to the oil phase at a volume ratio of 1:4 (aqueous phase:oil phase = 1:4), and homogenization was continued at high speed for 5 minutes to form an emulsion. Under continuous stirring at 400 rpm, the emulsion was added to a 60 vol% ethanol aqueous solution containing 2 wt% calcium chloride at three times the volume of the emulsion, and the reaction was carried out for 20 minutes to form solid microspheres. After standing, the precipitate was collected and washed three times each with anhydrous ethanol and diethyl ether, respectively. After vacuum drying at 40℃ for 8 hours, the mixture was passed through a 100-mesh sieve to obtain the active peptide-Terminalia chebula polyphenol complex loaded with Amanita tangutica mycelium.

[0057] The preparation method of Dendrobium nobile callus extract includes: taking sterile leaves of Dendrobium nobile seedlings, cutting them into small pieces, and placing them in MS liquid medium containing 1.5 mg / L 2,4-D and 0.2 mg / L 6-BA, and culturing them at 26℃ in the dark for 10 days to induce callus formation. The old medium is discarded, and new medium (the same medium as above) is added, and the mixture is cultured at 26℃ under light for 7 days. The callus is then collected and extracted twice by reflux at 80℃ using 80 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:10. Each extraction was performed for 2 hours. The extracts were combined, concentrated under reduced pressure to remove ethanol and some water, filtered through a 0.45 μm microporous membrane, and the filtrate was loaded onto an AB-8 macroporous resin column. The column was first washed with deionized water for 3 BV to remove impurities, and then eluted with 90 vol% ethanol aqueous solution for 4 BV. The eluent was collected and concentrated under reduced pressure at 60 °C to obtain an extract. Acetone with a mass twice that of the extract was added, and the column was allowed to stand at 6 °C for 18 hours to precipitate. The column was then centrifuged at 3000 r / min for 20 min, the precipitate was collected, and the column was dried under vacuum at 45 °C to obtain the Dendrobium nobile callus extract.

[0058] The preparation method of the gel matrix includes: taking konjac glucomannan, adding isopropanol at a mass ratio of 1:10, stirring and dispersing, adding 0.5% sodium hydroxide (catalyst) of konjac glucomannan by mass, adding propylene oxide at twice the mass of konjac glucomannan, reacting at 30℃ under nitrogen protection for 4h (hydroxypropylation modification), adding hydrochloric acid to a final concentration of 0.4mol / L to terminate the reaction, filtering, washing the precipitate three times with ethanol, and vacuum drying at 50℃ for 8h to obtain modified konjac glucomannan; mixing modified konjac glucomannan and carboxymethyl chitosan at a mass ratio of 2:1.8, and preparing a solution with purified water at a mass ratio of 1:6; adding 10% genipin of modified konjac glucomannan by mass, stirring and reacting at 40℃ for 30min; adding 2% EDTA-2Na of modified konjac glucomannan by mass, stirring for 10min, adjusting the viscosity to 5000mPa·s to obtain the gel matrix.

[0059] The preparation method of the above-mentioned active pharmaceutical ingredient for repairing oral mucosa includes the following steps:

[0060] The active peptides of Amanita fulva mycelium-Terminalia chebula polyphenol complex, Dendrobium nobile callus extract, sorbitol, menthol, gel matrix, and citric acid were added to deionized water, stirred and mixed evenly, and then vacuum degassed to form a gel.

[0061] Example 3

[0062] An active pharmaceutical ingredient for repairing oral mucosa, comprising the following raw materials in parts by weight: 8 parts of active peptide-Terminalia chebula polyphenol complex carrier, 6 parts of Dendrobium nobile callus extract, 3 parts of sorbitol, 0.3 parts of menthol, 25 parts of gel matrix, 0.3 parts of citric acid, and the balance being deionized water, totaling 100 parts.

[0063] The preparation method of active peptides from Amanita fulva mycelium includes: inoculating Amanita fulva into a liquid fermentation medium (formula: glucose 25 g / L, soybean meal 20 g / L, KH2PO4 1 g / L, MgSO4·7H2O) under aseptic conditions. 1.5 g / L (the remainder being water, pH at rest) was cultured aerobically at 25℃ and 180 rpm for 5 days to obtain mycelial culture medium. The mycelium was collected by centrifugation at 10000 rpm for 10 min, washed three times with physiological saline, and then diluted with deionized water to a mycelial suspension equal to 6 times the mycelial mass. The suspension was then homogenized by ultrasonication for 30 min at 500 W with a 2-second working cycle followed by a 5-second intermittent cycle. The pH was adjusted to 7.2, and the temperature to 50℃. Bromelain and Bacillus subtilis protease (both at 0.25% by weight) were added to the mycelial suspension, and the mixture was enzymatically hydrolyzed for 2 h. The enzymes were inactivated at 90℃ for 10 min, cooled, and centrifuged at 10000 rpm for 10 min. The supernatant was collected and ultrafiltered using an ultrafiltration membrane to obtain fractions between 1 kDa and 5 kDa. These fractions were then freeze-dried to obtain active peptides from Amanita fulva mycelium.

[0064] The preparation method of Terminalia chebula polyphenols includes: taking dried Terminalia chebula fruit, crushing it through a 40-mesh sieve, and extracting it twice at 80℃ for 2 hours each time with an 80 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:10. The extracts are combined, concentrated under reduced pressure to remove ethanol and some water, and then poured onto an AB-8 macroporous adsorption resin column. The column is first washed with deionized water for 3 BV to remove impurities, and then eluted with 70 vol% ethanol aqueous solution for 4 BV. The eluent is collected, concentrated under reduced pressure at 60℃, and dried under vacuum to obtain Terminalia chebula polyphenols.

[0065] The preparation method of sodium alginate-dopamine graft copolymer includes: preparing a 20 g / L sodium alginate solution with pH 5.5 MES buffer, adding 8 g / L EDC and 7 g / L NHS, and activating the carboxyl groups by stirring under nitrogen protection and in the dark at room temperature for 30 min to obtain an activated solution; dissolving dopamine hydrochloride in 3 times its mass of DMSO to obtain a dopamine hydrochloride solution; adding the dopamine hydrochloride solution to the activated solution at a mass ratio of sodium alginate:dopamine hydrochloride = 10:2.5 under nitrogen protection, in the dark, and at room temperature with stirring, and reacting for 24 h to obtain a reaction solution; adding the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzing with deionized water for 48 h, filtering through a 0.22 μm microporous membrane, and freeze-drying the filtrate to obtain the sodium alginate-dopamine graft copolymer.

[0066] The preparation method of the active peptide-Terminalia chebula polyphenol complex loading material from Amanita tangutica mycelium includes: mixing active peptides from Amanita tangutica mycelium and Terminalia chebula polyphenols at a mass ratio of 7:3 to obtain a complex; taking sodium alginate-dopamine graft copolymer, and mixing the complex and sodium alginate-dopamine graft copolymer together with deionized water at a mass ratio of total mass to water of 1:3 to prepare an internal aqueous phase; taking a 3wt%... Liquid paraffin from Span-80 was used as the oil phase. Under high-speed homogenization at 8000 rpm, the aqueous phase was added dropwise to the oil phase at a volume ratio of 1:6 (aqueous phase:oil phase = 1:6), and homogenization was continued at high speed for 3 minutes to form an emulsion. Under continuous stirring at 600 rpm, the emulsion was added to a 50 vol% ethanol aqueous solution containing 5 wt% calcium chloride at twice the volume of the emulsion, and the reaction was carried out for 40 minutes to form solid microspheres. After standing, the precipitate was collected and washed twice alternately with anhydrous ethanol and diethyl ether. After vacuum drying at 45℃ for 4 hours, the mixture was passed through a 120-mesh sieve to obtain the active peptide-Terminalia chebula polyphenol complex loaded with Amanita tangutica mycelium.

[0067] The preparation method of Dendrobium nobile callus extract includes: taking sterile leaves of Dendrobium nobile seedlings, cutting them into small pieces, and placing them in MS liquid medium containing 0.5 mg / L 2,4-D and 0.8 mg / L 6-BA, and culturing at 24℃ in the dark for 20 days to induce callus formation. The old medium is discarded, and new medium (the same as above) is added, and cultured at 24℃ under light for 10 days. The callus is then collected and extracted three times by reflux at 70℃ using a 70 vol% ethanol aqueous solution at a material-to-liquid mass ratio of 1:15. Each extraction was performed for 1 hour. The extracts were combined and concentrated under reduced pressure to remove ethanol and some water. After filtration through a 0.45 μm microporous membrane, the filtrate was loaded onto an AB-8 macroporous resin column. The column was first washed with deionized water to remove impurities (5 BV), and then eluted with 80 vol% ethanol aqueous solution (5 BV). The eluent was collected and concentrated under reduced pressure at 50 °C to obtain an extract. Four times the mass of the extract in acetone was added, and the column was allowed to stand at 4 °C for 16 hours to precipitate. The column was then centrifuged at 5000 r / min for 10 min, and the precipitate was collected and dried under vacuum at 55 °C to obtain the Dendrobium nobile callus extract.

[0068] The preparation method of the gel matrix includes: taking konjac glucomannan, adding isopropanol at a mass ratio of 1:8, stirring and dispersing, adding 1% sodium hydroxide (catalyst) of konjac glucomannan by mass, adding propylene oxide at a mass ratio of 1:1 that of konjac glucomannan, reacting at 50℃ under nitrogen protection for 2h (hydroxypropylation modification), adding hydrochloric acid to a final concentration of 0.6mol / L to terminate the reaction, filtering, washing the precipitate twice with ethanol, and vacuum drying at 70℃ for 4h to obtain modified konjac glucomannan; mixing modified konjac glucomannan and carboxymethyl chitosan at a mass ratio of 3:1.4, and preparing a solution with purified water at a mass ratio of 1:8; adding 8% genipin of modified konjac glucomannan by mass, stirring and reacting at 50℃ for 20min; adding 3% EDTA-2Na of modified konjac glucomannan by mass, stirring for 5min, and adjusting the viscosity to 8000mPa·s to obtain the gel matrix.

[0069] The preparation method of the above-mentioned active pharmaceutical ingredient for repairing oral mucosa includes the following steps:

[0070] The active peptides of Amanita fulva mycelium-Terminalia chebula polyphenol complex, Dendrobium nobile callus extract, sorbitol, menthol, gel matrix, and citric acid were added to deionized water, stirred and mixed evenly, and then vacuum degassed to form a gel.

[0071] Comparative Example 1

[0072] The difference from Example 1 is that the active peptide-Terminalia chebula polyphenol complex loading material of Amanita tangutica mycelium was modified to 4 parts, and the Dendrobium nobile callus extract was modified to 10 parts.

[0073] Comparative Example 2

[0074] The difference from Example 1 is that in the preparation method of the active peptides of Amanita fulva mycelium, bromelain is replaced with papain.

[0075] Comparative Example 3

[0076] The difference from Example 1 is that in the preparation method of Amanita fulva mycelium active peptides, Bacillus subtilis protease is replaced with papain.

[0077] Comparative Example 4

[0078] The difference from Example 1 is that Bacillus subtilis protease is not added in the preparation method of the active peptides of Amanita fulva mycelium.

[0079] Comparative Example 5

[0080] The difference from Example 1 is that Terminalia chebula polyphenols are not prepared, and Terminalia chebula polyphenols are not added to the Amanita fulva mycelium active peptide-Terminia chebula polyphenol complex.

[0081] Comparative Example 6

[0082] The difference from Example 1 is that: sodium alginate-dopamine graft copolymer is not prepared, and the active peptide-Terminalia chebula polyphenol composite loading material is directly replaced by a mixture of active peptide and Terminalia chebula polyphenol; that is, no loading is performed.

[0083] Comparative Example 7

[0084] The difference from Example 1 is that: Dendrobium nobile callus extract is directly replaced by Dendrobium nobile extract; in the preparation method of Dendrobium nobile callus extract, callus tissue is not prepared, and the callus tissue is directly made from Dendrobium nobile leaf powder.

[0085] Comparative Example 8

[0086] The difference from Example 1 is that in the preparation of Dendrobium nobile callus extract, the AB-8 macroporous resin column is replaced with S-8 macroporous adsorption resin.

[0087] Comparative Example 9

[0088] The difference from Example 1 is that konjac glucomannan is not hydroxypropylated in the preparation of the gel matrix.

[0089] The raw materials used in the above embodiments and comparative examples are sourced as follows: Sorbitol from Shandong Yunding Chemical Co., Ltd.; Menthol from Jiangxi Tianshihe Pharmaceutical Co., Ltd.; Citric acid from Qingdao Haiweisen Biotechnology Co., Ltd.; Bromelain from Shanxi Zhongnuo Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g; Bacillus subtilis protease from Hebei Runying Biotechnology Co., Ltd., with an enzyme activity of 200,000 U / g; AB-8 macroporous adsorption resin from Shanghai Yuanye Biotechnology Co., Ltd.; Sodium alginate from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.; EDC from Hubei Hengjingrui Chemical Co., Ltd.; NHS from Shanghai Lanrun Chemical Co., Ltd.; Dopamine hydrochloride from Xi'an Pinjian Biotechnology Co., Ltd.; DMSO from Nanjing Chemical Reagent Co., Ltd.; Span-80 from Shandong Tiandao Bioengineering Co., Ltd.; Liquid paraffin from Shanxi Jinyang Pharmaceutical Excipients Co., Ltd.; 2,4-D from Shanghai Youpeng Chemical Co., Ltd.; 6-BA from Hubei Rishengchang New Material Technology Co., Ltd.; MS culture medium from Qingdao Qingyao Bioengineering Co., Ltd.; Konjac glucomannan from Xi'an Zebang Biotechnology Co., Ltd.; and Isopropanol from Nanjing Chemical Reagent Co., Ltd. Propylene oxide was sourced from Shandong Feier New Materials Co., Ltd. Carboxymethyl chitosan was sourced from Qingdao Boyite Biomaterials Co., Ltd. Genipin was sourced from Nanjing Puyan Biotechnology Co., Ltd. EDTA-2Na was sourced from Xi'an Jinxiang Pharmaceutical Excipients Co., Ltd. Papain was sourced from Hebei AsiaInfo Biotechnology Co., Ltd., with an enzyme activity of 200,000 U / g. S-8 macroporous adsorption resin was sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0090] I. Detection of oral mucosal epithelial cell proliferation:

[0091] Experimental subjects: the active pharmaceutical ingredients of Examples 1 to 3 and Comparative Examples 1 to 8 (the active pharmaceutical ingredients refer to the active peptide-Terminalia chebula polyphenol complex carrier and Dendrobium nobile callus extract, without flavoring agents and gelling agents or other excipients).

[0092] Sample preparation: For the examples and comparative examples, the active peptide-Terminalia chebula polyphenol complex of Amanita tangutica mycelium and Dendrobium nobile callus extract were mixed according to their mass fraction in the drug and prepared into a working concentration sample of 250 μg / mL using DMEM medium, i.e. drug-containing medium. The sample was then filtered through a 0.22 μm filter membrane for sterilization.

[0093] The detection method included: culturing human immortalized oral mucosal epithelial cells (HIOEC) and seeding them in 96-well plates at 5 × 10⁶ cells / well. 3Cells were cultured in wells for 24 hours until adherence, then the medium was replaced with drug-containing medium. The control group used standard DMEM medium. Each group had 5 replicates and was cultured for 72 hours. The MTT assay was performed: 20 μL of MTT solution (5 mg / mL dissolved in PBS) was added to each well, and the cells were incubated at 37°C for 4 hours. The supernatant was discarded, and 150 μL of LDMSO was added to each well. The cells were shaken for 10 minutes, and the absorbance (OD) at 490 nm was measured using a microplate reader. Cell proliferation rate (%) = (OD value of experimental group / OD value of control group) × 100%.

[0094] II. Wound Healing Model Detection:

[0095] Experimental subjects: Same as Experiment 1.

[0096] Sample preparation: Same as the drug-containing culture medium in Experiment 1.

[0097] The detection method included: seeding HIOEC cells into 6-well plates at 2 × 10⁶ cells / well. 5 Cells were cultured in wells until confluence reached 90% or higher. A sterile 200 μL pipette tip was used to make a straight incision on the bottom of each well. Exfoliated cells were washed away with PBS. The experimental group received drug-containing medium, while the control group received standard DMEM medium. Each group was divided into three replicates. Cells were incubated at 37°C and 5% CO2 for 72 h. The incised area was observed using an inverted microscope (×100). The incised width was measured using ImageJ software, and the healing rate was calculated. Healing rate (%) = (0 h incised width - post-culture incised width) / 0 h incised width × 100%.

[0098] III. Detection of inflammatory factor expression:

[0099] Experimental subjects: Same as Experiment 1.

[0100] Group preparation: HIOEC cells were seeded at a rate of 2 × 10⁻⁶. 5 Cells were seeded per well in 6-well plates and cultured for 24 hours until adherence. The medium was then replaced with DMEM containing 1 μg / mL LPS for the inflammation model group, DMEM containing 1 μg / mL LPS and 250 μg / mL of the active pharmaceutical ingredient for the experimental group, and DMEM containing neither LPS nor the drug for the normal control group (to verify the induction effect of the inflammation model group). Cell supernatant was collected after another 24 hours of culture. Each group was divided into three replicates.

[0101] The detection method included: following the instructions for the ELISA kits for human IL-6 and TNF-α, measuring the OD value at a wavelength of 450 nm using a microplate reader. A standard curve was plotted based on the OD values ​​of the standards, and the concentrations of IL-6 and TNF-α in the experimental group were calculated and compared with those in the inflammation model group. The inflammatory factor inhibition rate (%) was calculated as: [(Inflammation model group concentration - Experimental group concentration) / Inflammation model group concentration] × 100%.

[0102] IV. Collagen Synthesis Detection:

[0103] Experimental subjects: Same as Experiment 1.

[0104] Group preparation: Cultured human skin fibroblasts (HSF), 2×10 5 Cells were seeded per well in 6-well plates and cultured until adherent. Then, the same drug-containing medium as in Experiment 1 was added, and the cells were cultured for 48 hours. A blank control group (using standard DMEM medium) was also included, and cultured for 48 hours. Each group had 3 replicates.

[0105] The detection method included: discarding the old culture medium, washing cells twice with PBS, fixing with 4% (w / v) paraformaldehyde for 30 min, washing with PBS, adding 0.1% Sirius Red staining solution (dissolved in saturated picric acid), and staining at room temperature for 1 h. Washing three times with 0.01 mol / L hydrochloric acid to remove unbound stain, adding 1 mL of 0.1 mol / L NaOH solution, shaking for 10 min to dissolve the dye, and measuring the OD value at 540 nm using a microplate reader. The relative collagen content (%) = (OD value of experimental group / OD value of blank group) × 100%.

[0106] V. Adhesion Duration Test:

[0107] Experimental subjects: the gel product of Example 1 (containing all ingredients); the gel product of Comparative Example 9 (containing all ingredients).

[0108] Sample preparation: The gel was evenly applied to the surface of the porcine oral mucosa (area 1cm×1cm, thickness 0.5mm), and placed in phosphate buffer at 37℃ and pH 6.8. At the same time, a reciprocating shaker was used to simulate chewing action (frequency 60 times / min, amplitude 5mm).

[0109] The detection methods included: observing the residue of the gel on the mucosal surface every 10 minutes, calculating the percentage of the residual area to the initial area using ImageJ software, recording the time when the residual area was <50% (i.e., the adhesion duration), and taking the average value of 3 parallel samples per group.

[0110] Table 1. Test Results (Average Values)

[0111]

[0112] Note: "-" indicates that the corresponding indicator was not tested in this group (Comparative Examples 1-8 only tested indicators related to the active ingredient and did not include gel excipients; Comparative Example 9 only tested gel adhesion properties and did not involve indicators of the active ingredient). Among them, the scratch healing rate of the blank control group tested in the wound healing model was 22.5%.

[0113] In Examples 1 to 3, the active peptides from *Amanita fulva* mycelium and the polyphenols from *Terminalia chebula* were appropriately matched with the callus extract from *Dendrobium nobile*. These active peptides, polyphenols, and the callus extract exhibited excellent synergistic effects, covering the entire process of proliferation, anti-inflammation, and remodeling repair, resulting in enhanced efficacy. The sodium alginate-dopamine graft copolymer, which encapsulates the active ingredients, is more stable, preventing degradation and oxidation, reducing the loss of active ingredients, and enabling slow release to ensure sustained action. It is also gentle on cells and reduces toxicity. Key process improvements include: synergistic enzymatic hydrolysis by bromelain and Bacillus subtilis protease to produce 1-5 kDa characteristic active peptides; and induction of callus tissue by 2,4-D and 6-BA in *Dendrobium nobile* to promote the generation of effective components, including active polysaccharides and alkaloids, thereby enhancing the efficacy of active substances. Hydroxypropyl-modified konjac glucomannan and carboxymethyl chitosan form a highly adhesive gel that resists friction, prevents loss of active ingredients, and provides a stable and moist environment for repair.

[0114] Comparative Example 1 reduced the amount of *Amanita fulva* mycelium active peptide-Terminalia chebula polyphenol complex loading to 4 parts and increased the amount of *Dendrobium nobile* callus extract to 10 parts, disrupting the balance of proliferation, anti-inflammation, and remodeling. *Amanita fulva* mycelium active peptides are key components for activating epithelial cell division; their reduced content directly led to a decrease in the rate of epithelial cell transition from G1 to S phase, significantly reducing cell proliferation. Simultaneously, the regulatory effect of active peptides on epithelial cell migration-related proteins weakened, resulting in a decrease in scratch healing rate. Negative interference from excessive extracts: Although *Dendrobium nobile* callus extract contains anti-inflammatory components, excessive amounts introduce some water-soluble impurities that compete with active peptides for binding to cell surface receptors, further inhibiting proliferation signal transduction. *Dendrobium nobile* callus extract cannot compensate for the lack of active peptides in repair, ultimately resulting in lower inflammatory factor inhibition rates and lower relative collagen content compared to the example.

[0115] The improper selection and combination of proteases in Comparative Examples 2 to 4 reduced the quality of the active peptides. Enzyme deficiency or substitution led to changes in enzymatic hydrolysis specificity, different protein cleavage sites, and ultimately different peptide chain structures, cell utilization rates, and binding to different cell receptors, resulting in varying effects. Furthermore, the introduced impurity peptides also affected safety, causing different degrees of change in various indicators. This demonstrates that the peptide products prepared by the protease combination in Example 1 are more effective in mucosal repair and safety.

[0116] Comparative Example 5 showed that the absence of Terminalia chebula polyphenols resulted in the loss of synergistic effects between proliferation and protection, disrupting the synergistic mechanism between the bioactive peptides promoting proliferation and the Terminalia chebula polyphenols protecting the microenvironment. Terminalia chebula polyphenols contain various antioxidants that scavenge ROS in wounds and reduce ROS-induced oxidative damage to epithelial cell DNA; their absence leads to ROS accumulation, resulting in increased apoptosis rates and directly reducing cell proliferation. Components in Terminalia chebula polyphenols can directly inhibit the release of IL-6 and TNF-α from inflammatory cells, complementing the mechanism by which bioactive peptides inhibit inflammation through signaling pathways; their absence results in insufficient anti-inflammatory capacity of individual bioactive peptides, worsening the inflammatory microenvironment in the wound, affecting both epithelial cell migration and inhibiting the efficiency of fibroblast collagen synthesis.

[0117] Comparative Example 6 did not prepare the sodium alginate-dopamine graft copolymer, but directly used a mixture of active peptides and polyphenols. This resulted in the loss of the protective and controlled-release functions of the encapsulation system, affecting the stability of the active ingredients. The protein peptides were easily hydrolyzed by enzymes, and the polyphenols were easily oxidized. Without controlled-release, the gradual absorption performance was lost, affecting cellular absorption regulation. Some harmful impurities were released at once, stimulating cells and delaying the repair process. The ease with which protein peptides are hydrolyzed by enzymes and polyphenols are oxidized ultimately affects the overall efficacy.

[0118] Comparative Example 7 used Dendrobium nobile leaf powder extract instead of callus extract, resulting in differences in the types, content, and purity of active ingredients. Callus tissue is a dedifferentiated pluripotent cell mass that, under the regulation of 2,4-D and 6-BA, can directionally accumulate various effective repair components, including polysaccharides and alkaloids, which can directly promote fibroblast activation. Natural leaves contain more impurities such as cellulose and tannins, resulting in low levels of repair-active substances. Furthermore, the extraction process easily introduces inhibitory components. These impurities compete for binding to polysaccharide receptors on the surface of fibroblasts, reducing collagen synthesis efficiency and weakening the effects of anti-inflammatory components.

[0119] In Comparative Example 8, replacing AB-8 macroporous adsorption resin with S-8 macroporous adsorption resin resulted in a decrease in the purification effect of Dendrobium nobile callus extract. S-8 and AB-8 have different adsorption specificities. S-8 led to differences in the types and purity of the final purified components, and introduced impurities that competed with the effective active ingredients for binding to cell receptors, interfering with the absorption of effective components by cells, and affecting various indicators.

[0120] Comparative Example 9, without hydroxypropylation modification of konjac glucomannan, resulted in insufficient adhesion performance of the gel matrix. The mechanism of modification: Hydroxypropylation introduces hydroxypropyl groups into the konjac glucomannan molecular chain, reducing intermolecular hydrogen bonding and improving the gel's flexibility and hydrophilicity. Simultaneously, it enhances the hydrophobic interaction with mucosal surface mucins, forming a stable gel-mucosal binding layer. Unmodified konjac glucomannan molecular chains are rigid and easily break under simulated chewing oscillations, failing to form a sustained adhesion layer.

Claims

1. An active pharmaceutical ingredient for repairing oral mucosa, characterized in that, The medicine is composed of the following components in parts by mass: orange gill tricholoma mycelium active peptide- Terminalia chebula polyphenol complex encapsulation 8-10 parts, dendrobium stem leaf callus extract 4-6 parts, sorbitol 3-5 parts, menthol 0.1-0.3 parts, gel matrix 25-35 parts, citric acid 0.1-0.3 parts, and the rest deionized water, with the total amount being 100 parts; The orange gill tricholoma mycelium active peptide- Terminalia chebula polyphenol complex encapsulation is obtained by graft copolymerization of sodium alginate-dopamine, cross-linking and solidification of the complex of orange gill tricholoma mycelium active peptide and Terminalia chebula polyphenol; the mass ratio of the orange gill tricholoma mycelium active peptide and the Terminalia chebula polyphenol is (6-7):(3-4); The orange gill tricholoma mycelium active peptide is a product of 1-5 kDa obtained by co-enzymolysis of orange gill tricholoma mycelium with bromelain and bacillus subtilis protease; the Terminalia chebula polyphenol is obtained by extracting Terminalia chebula fruits with an aqueous ethanol solution and purifying the extract with an AB-8 macroporous adsorption resin column; the sodium alginate-dopamine graft copolymer is obtained by reacting sodium alginate activated with EDC and NHS with hydrochloric acid dopamine; The dendrobium stem leaf callus extract is obtained by sequentially inducing the sections of dendrobium stem leaf in MS liquid medium with 2,4-D and 6-BA, culturing in the dark and culturing in the light, extracting the obtained callus with an aqueous ethanol solution, adsorbing the extract with an AB-8 macroporous resin column, and obtaining the precipitate by standing the eluate in acetone; The viscosity of the gel matrix is 5000-8000 mPa·s; the gel matrix is obtained by reacting modified konjac glucomannan, carboxymethyl chitosan, genipin and EDTA-2Na; the mass ratio of the modified konjac glucomannan and the carboxymethyl chitosan is (2-3):(1.4-1.8); the modified konjac glucomannan is obtained by hydroxypropylation of konjac glucomannan with propylene oxide.

2. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1, characterized in that, The preparation method of the orange gill tricholoma mycelium active peptide- Terminalia chebula polyphenol complex encapsulation comprises the following steps: mixing orange gill tricholoma mycelium active peptide and Terminalia chebula polyphenol according to the mass ratio (6-7):(3-4) to obtain a complex; preparing an internal water phase by mixing the complex and sodium alginate-dopamine graft copolymer according to the mass ratio of 1:(1.2-1.5); taking liquid paraffin containing 1-3 wt% Span-80 as an oil phase; under high-speed homogenization at 8000-10000 rpm, dropping the internal water phase into the oil phase, continuously homogenizing to form an emulsion; under continuous stirring at 400-600 rpm, adding the emulsion into 50-60 vol% aqueous ethanol solution containing 2-5 wt% calcium chloride, reacting to form solid microspheres; standing, washing the precipitate with anhydrous ethanol and diethyl ether alternately, vacuum drying, sieving, and obtaining the orange gill tricholoma mycelium active peptide- Terminalia chebula polyphenol complex encapsulation.

3. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1 or 2, characterized in that, The preparation method of the active peptide of the orange-capped tricholoma mycelium comprises the following steps: washing the mycelium of the orange-capped tricholoma with normal saline, then diluting the mycelium with deionized water to obtain a mycelium suspension, and homogenizing the mycelium suspension by ultrasonic crushing; adjusting pH to 6.5-7.2 and temperature to 50-55 DEG C, adding bromelain and bacillus subtilis protease, and performing enzymolysis for 2-3 hours; inactivating the enzyme, cooling, centrifuging, taking the supernatant, ultrafiltering the supernatant with an ultrafiltration membrane, obtaining a component with a molecular weight of 1-5 kDa, and freeze-drying to obtain the active peptide of the orange-capped tricholoma mycelium.

4. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 3, characterized in that, The mycelium is obtained by culturing the orange-capped tricholoma in a liquid fermentation medium; the adding amount of the bromelain and the bacillus subtilis protease is 0.15-0.25% of the mass of the mycelium suspension; the enzyme inactivation is performed at 85-90 DEG C for 10-15 minutes; and the centrifugation is performed at 9000-10000 rpm for 10-15 minutes.

5. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1 or 2, characterized in that, The preparation method of the Terminalia chebula polyphenol comprises the following steps: crushing dried Terminalia chebula fruits, reflux extracting the crushed Terminalia chebula fruits with 70-80 vol% ethanol aqueous solution at 70-80 DEG C, concentrating the extraction liquid under reduced pressure, eluting the concentrated extraction liquid on an AB-8 macroporous adsorption resin column, first washing the column with deionized water for 3-5 BV to remove impurities, then eluting the column with 60-70 vol% ethanol aqueous solution for 4-5 BV, collecting the eluate, concentrating the eluate under reduced pressure, and vacuum drying to obtain the Terminalia chebula polyphenol.

6. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1 or 2, characterized in that, The preparation method of the sodium alginate-dopamine graft copolymer comprises the following steps: preparing a 15-20 g / L sodium alginate solution by using a MES buffer solution with pH of 5.5-6.5, adding 8-12 g / L EDC and 5-7 g / L NHS, activating carboxyl groups under stirring at room temperature in a nitrogen atmosphere and in the dark to obtain an activation liquid; dissolving hydrochloric acid dopamine in 2-3 times the mass of DMSO to obtain a hydrochloric acid dopamine solution; adding the hydrochloric acid dopamine solution into the activation liquid in a mass ratio of sodium alginate: hydrochloric acid dopamine = 10: (2.5-3.5) under stirring at room temperature in a nitrogen atmosphere and in the dark, and reacting to obtain a reaction liquid; placing the reaction liquid in a dialysis bag with a molecular weight cut-off of 3500 Da, dialyzing the reaction liquid with deionized water, filtering the dialyzed reaction liquid, and freeze-drying the filtrate to obtain the sodium alginate-dopamine graft copolymer.

7. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1, characterized in that, The preparation method of the Dendrobium devonianum callus extract includes the following steps: taking Dendrobium devonianum sterile seedling leaves, cutting into small pieces, placing in MS liquid medium containing 0.5 mg / L-1.5 mg / L 2,4-D and 0.2 mg / L-0.8 mg / L 6-BA, culturing at 24-26 DEG C in the dark for 10-20 days to induce callus formation, replacing the new culture medium, culturing at 24-26 DEG C under light for 7-10 days; taking the callus, refluxing with 70vol%-80vol% ethanol aqueous solution at 70-80 DEG C, reducing pressure and concentrating, filtering, loading the filtrate onto an AB-8 macroporous resin column, first washing with 3BV-5BV deionized water to remove impurities, then eluting with 80vol%-90vol% ethanol aqueous solution for 4BV-5BV, collecting the eluate, reducing pressure and concentrating to extract, adding acetone and standing for precipitation, centrifuging, collecting the precipitate, and vacuum drying to obtain the Dendrobium devonianum callus extract.

8. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 1, characterized in that, The preparation method of the gel matrix includes the following steps: mixing modified konjac glucomannan and carboxymethyl chitosan according to a mass ratio of (2-3):(1.4-1.8), preparing a solution with purified water, adding 8%-10% of genipin based on the mass of the modified konjac glucomannan, stirring and reacting, adding 2%-3% of EDTA-2Na based on the mass of the modified konjac glucomannan, stirring, and adjusting the viscosity to 5000-8000 mPa·s to obtain the gel matrix.

9. An active pharmaceutical ingredient for the repair of oral mucosa according to claim 8, characterized in that, The preparation method of the modified konjac glucomannan includes the following steps: adding isopropyl alcohol to konjac glucomannan, stirring and dispersing, adding 0.5%-1% of sodium hydroxide based on the mass of the konjac glucomannan, adding 1-2 times of propylene oxide based on the mass of the konjac glucomannan, reacting, adding hydrochloric acid to terminate the reaction, filtering, washing the precipitate with ethanol, and vacuum drying to obtain the modified konjac glucomannan.

10. The method of claim 1, wherein the active pharmaceutical ingredient is prepared by the steps of: The method comprises the following steps: According to mass fractions, the orange-capped tricholoma mycelial active peptide-fructus terminaliae polyphenol complex encapsulating material, the Dendrobium devonianum callus extract, sorbitol, menthol, the gel matrix, and citric acid are added into deionized water, stirred and mixed uniformly, vacuum degassed, and a gel is formed.

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