A toothpaste composition for improving gum bleeding and a method for preparing the same

Abstract

The application belongs to the technical field of oral care products, and discloses a toothpaste composition for improving gum bleeding and a preparation method thereof. The composition is composed of targeted anti-inflammatory nanospheres, barrier repair composite agents, inactivated probiotic Lactobacillus plantarum and toothpaste basic auxiliary materials. The targeted anti-inflammatory nanospheres are hydroxypropyl-beta-cyclodextrin microspheres with ICAM-1 targeting peptides grafted on the surface, and the inside is embedded with active substances compounded from white wine and epigallocatechin gallate; the barrier repair composite agents are compounded from recombinant humanized collagen type III and sodium hyaluronate. The mild auxiliary material system without SLS can precisely enrich the active substances at the inflammation site of the gums, inhibit the inflammatory response, repair the gum epithelial barrier, regulate the balance of the oral microecology, effectively improve the gum bleeding and reduce the recurrence rate, and the active ingredients have good stability, low mucous membrane irritation and are suitable for long-term use of the vulnerable population of gums.

Description

Technical Field

[0001] This invention belongs to the field of oral care products technology, specifically relating to a toothpaste composition for improving gingival bleeding and its preparation method. Background Technology

[0002] Bleeding gums are the most common early symptom of periodontal disease, which can develop into chronic periodontitis, eventually leading to gingival recession, tooth loosening, and even tooth loss. The core pathogenesis of bleeding gums involves three levels: First, pathogenic bacteria such as Porphyromonas gingivalis in dental plaque colonize and multiply, inducing a local inflammatory response, activating the NF-κB signaling pathway, and promoting the release of large amounts of pro-inflammatory factors such as IL-6 and TNF-α, resulting in gingival capillary dilation and increased permeability, making bleeding easily triggered by even slight mechanical stimulation; second, long-term inflammation damages the gingival epithelial barrier, disrupting the tight junctions between epithelial cells, making it easier for external stimuli and pathogenic bacteria to invade deeper tissues; third, periodontal microecological imbalance, with a decrease in the abundance of beneficial bacteria and an increase in the proportion of pathogenic bacteria, reduces the innate immunity of the oral mucosa, leading to recurrent inflammation and persistent bleeding gums.

[0003] Currently, commercially available hemostatic toothpastes mainly fall into two categories: one type contains hemostatic / coagulant ingredients such as tranexamic acid and Yunnan Baiyao extract, which can only promote blood clotting and relieve bleeding symptoms in the short term, but cannot inhibit the root cause of inflammation or repair damaged gum tissue. Relapse is very likely after discontinuation of the medication, and long-term use of hemostatic ingredients poses potential safety risks. The other type contains natural plant extracts such as tea polyphenols and resveratrol, which have both anti-inflammatory and antibacterial effects. However, these ingredients have poor water solubility and insufficient stability, short residence time in the oral cavity, low bioavailability, and lack lesion targeting. Most of the active ingredients diffuse throughout the oral cavity and are diluted by saliva, resulting in a very low concentration of drug that is actually concentrated at the site of gum inflammation, making it difficult to achieve the expected efficacy.

[0004] In addition, most toothpastes currently available contain highly irritating anionic surfactants such as sodium lauryl sulfate (SLS) to enhance cleaning power and foaming ability. SLS can damage the cell membranes of gingival epithelial cells, exacerbating gingival sensitivity and barrier damage. For gums that are already inflamed and damaged, it can intensify stinging sensations and inflammatory responses, which is actually detrimental to improving gingival bleeding. Some products add broad-spectrum antibacterial agents such as triclosan to enhance antibacterial effects. While inhibiting pathogenic bacteria, triclosan can also kill beneficial bacteria in the oral cavity, disrupting the microecological balance. Long-term use can actually reduce the oral cavity's own immunity and increase the probability of recurrent inflammation. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a toothpaste composition for improving gingival bleeding and its preparation method. With "treating both the symptoms and the root cause, and providing long-lasting repair" as its core, it constructs three core functional units: a "targeted anti-inflammatory and hemostatic unit," a "gingival barrier repair unit," and a "periodontal microecological regulation unit." Combined with a mild excipient system free of SLS, it achieves multiple effects such as rapidly relieving gingival bleeding, providing long-lasting repair of the gingival barrier, and reducing inflammation recurrence from the root cause. At the same time, it has good biocompatibility and activity stability.

[0006] The objective of this invention can be achieved through the following technical solutions: A toothpaste composition for improving gingival bleeding, comprising, by weight, the following components: 0.5-5 parts targeted anti-inflammatory nanospheres, 0.2-3 parts barrier repair complex, 0.1-2 parts inactivated Lactobacillus plantarum biotic, and 85-98 parts toothpaste base excipients.

[0007] As a preferred technical solution of the present invention, the targeted anti-inflammatory nanospheres are hydroxypropyl-β-cyclodextrin nanospheres with ICAM-1 targeting peptides covalently grafted on their surface, and the nanospheres contain a complex active ingredient of resveratrol and epigallocatechin gallate in a mass ratio of 1:1 to 1:3. The barrier repair compound is composed of recombinant type III humanized collagen and oligomeric sodium hyaluronate in a mass ratio of 1:1 to 1:5.

[0008] As a preferred embodiment of the present invention, the total drug loading of resveratrol and epigallocatechin gallate in the targeted anti-inflammatory nanospheres is 8%-15%, and the encapsulation efficiency is ≥85%; the grafting amount of the ICAM-1 targeted peptide is 1.5%-3% of the mass of the microspheres.

[0009] As a preferred embodiment of the present invention, the average particle size of the targeted anti-inflammatory nanospheres is 150-300 nm.

[0010] As a preferred technical solution of the present invention, the inactivated Lactobacillus plantarum biogener is obtained by fermenting Lactobacillus plantarum, inactivating it at high temperature, and freeze-drying it, wherein the bacterial cell content is ≥1×10¹¹CFU / g, and it includes intact bacterial cell fragments and extracellular metabolites.

[0011] As a preferred embodiment of the present invention, the toothpaste base excipients, by weight, consist of the following components: 20-40 parts of abrasive, 20-35 parts of humectant, 1-5 parts of amino acid surfactant, 0.5-2 parts of thickener, 0.1-0.5 parts of sweetener, 0.5-1.5 parts of edible flavoring, and purified water to make up to 100 parts.

[0012] As a preferred embodiment of the present invention, the friction agent is one or both of hydrated silica and aluminum hydroxide; The moisturizer is at least two of glycerin, sorbitol, and polyethylene glycol-400; The surfactant is one or two of sodium lauroyl sarcosinate and potassium cocoyl glycinate. The thickener is at least two of sodium carboxymethyl cellulose, xanthan gum, and carbomer.

[0013] A method for preparing the above-mentioned toothpaste composition for improving gingival bleeding includes the following steps: S1: Hydroxypropyl-β-cyclodextrin was dissolved in purified water, and an ethanol solution of resveratrol and epigallocatechin gallate was added. After constant temperature stirring and encapsulation, the mixture was spray-dried to obtain nanosphere powder. The nanosphere powder was activated by EDC / NHS, and ICAM-1 targeting peptide was added for covalent grafting. After dialysis purification and freeze-drying, targeted anti-inflammatory nanospheres were obtained. S2: Add humectant, thickener and sweetener to purified water, stir until completely swollen, heat to 45-55℃ and then add abrasive, disperse at high speed until uniform and free of particles to obtain aqueous matrix; S3: Add the barrier repair complex and inactivated Lactobacillus plantarum biogen to purified water at 4°C and stir at low speed until completely dissolved and dispersed to obtain an active premix; S4: Cool the aqueous matrix to 25-30℃, add the surfactant and stir at low speed until completely dissolved, then add the targeted anti-inflammatory nanospheres and disperse evenly; then slowly add the active premixed liquid, and after the addition is complete, vacuum degas for 15-30 minutes, add the edible flavoring and continue to homogenize and stir to obtain the toothpaste composition for improving gingival bleeding.

[0014] As a preferred embodiment of the present invention, in step S1, the inclusion reaction temperature is 35-45℃, the stirring rate is 300-500rpm, and the inclusion time is 2-4h; the pH of the targeted peptide grafting reaction system is 5.5-6.5, the reaction temperature is 2-8℃, and the reaction time is 12-18h.

[0015] Application of the toothpaste composition for improving gingival bleeding as described above in the preparation of oral care products that improve gingival bleeding, repair the gingival epithelial barrier, and regulate the periodontal microecology. The beneficial effects of this invention are: 1. Nanospheres modified with ICAM-1 targeting peptides precisely deliver active ingredients to gingival inflammation sites, effectively increasing the drug concentration at the lesion site compared to free active ingredients. This rapidly inhibits the NF-κB inflammatory pathway, reduces capillary permeability, and significantly improves gingival bleeding symptoms. The synergistic effect of resveratrol and EGCG is superior to that of a single component in terms of anti-inflammatory and antibacterial effects, without disrupting the balance of oral flora.

[0016] 2. Recombinant type III humanized collagen and oligomeric sodium hyaluronate work synergistically to repair the gingival epithelial barrier and connective tissue, enhance the gums' resistance to external stimuli, and block the vicious cycle of "pathogenic bacteria invasion - inflammation - barrier damage" to achieve long-term repair.

[0017] 3. After inactivation, Lactobacillus plantarum regulates the periodontal microecological balance, enhances the innate immunity of the oral mucosa, reduces the frequency of inflammation from the root cause of the disease, and has a low recurrence rate after discontinuation of the drug, truly achieving both symptomatic and radical treatment.

[0018] 4. The formula is free of SLS, broad-spectrum antibacterial agents, and procoagulant drugs, and has extremely low irritation to the oral mucosa. It will not aggravate gum sensitivity and is suitable for long-term use by sensitive people with fragile gums and easy bleeding.

[0019] 5. Cyclodextrin inclusion technology effectively protects resveratrol and EGCG from oxidative degradation, and the inactivated glycogen and collagen exhibit excellent stability in a mild excipient system. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with embodiments, is provided below.

[0021] Hydroxypropyl-β-cyclodextrin: Xi'an Xihai Biotechnology Co., Ltd.; Resveratrol: Xi'an Tianfeng Biotechnology Co., Ltd.; EGCG: Guangdong Mingtong Biotechnology Co., Ltd.; ICAM-1: Thermo Fisher RP-75643; Recombinant Type III Humanized Collagen: Hubei Darli Chemical Co., Ltd.; Oligomeric sodium hyaluronate: Bloomage Biotechnology Co., Ltd.; Lactobacillus plantarum inactivated biogen: prepared by fermentation and inactivation of Lactobacillus plantarum MICROBIOLOGICS®01144K, with a viable count ≥1×10¹¹CFU / g before inactivation, self-made; Hydrated silica: Guangzhou Dinghao Chemical Co., Ltd.; All other excipients are commercially available toothpaste-grade products.

[0022] Preparation of biogenic agents after inactivation of Lactobacillus plantarum: The inactivated Lactobacillus plantarum biogen is prepared from Lactobacillus plantarum as the starting strain through a series of activation steps, liquid deep fermentation, high-temperature inactivation, and co-drying of bacterial cells and metabolites. It completely retains the cell wall components and extracellular metabolic active substances. The specific preparation steps are as follows: Strain activation: Take Lactobacillus plantarum strain, thaw at room temperature, and inoculate into sterile MRS liquid medium at an inoculation rate of 2% (v / v). Incubate anaerobically at 37°C for 20 h. Activate by subculturing twice until the bacterial culture reaches OD. 60n The value was stabilized at 1.8~2.0, resulting in a working bacterial solution with uniform viability.

[0023] Seed culture expansion: The activated working bacterial solution was inoculated into the seed culture medium at a rate of 3% (v / v). The seed culture medium consisted of the following components by weight and volume: glucose 20 g / L, peptone 10 g / L, beef extract powder 8 g / L, yeast extract 4 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, anhydrous sodium acetate 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.05 g / L, Tween-80 1 mL / L, and natural pH. The culture was anaerobic at 37℃ and 100 rpm for 12 h until the late logarithmic growth phase of the bacterial cells, which was then the primary seed culture.

[0024] Batch fermentation in a 50L fermenter: Primary seed culture was inoculated into the fermentation medium at a 5% (v / v) inoculation rate. The fermentation medium had the same composition as the seed culture medium, with a volume of 30L. The fermentation temperature was maintained at a constant 37℃, and the stirring speed was 80 rpm. Sterile nitrogen was purged throughout the process to maintain an anaerobic environment inside the tank. During fermentation, the pH was adjusted online using 2 mol / L sodium hydroxide solution to maintain the system pH at 6.0 ± 0.2. After 16–18 hours of fermentation, samples were taken and viable cell counts were determined using the plate count method. When the viable cell count reached 1.2 × 10⁻⁶... 9 Fermentation is terminated when the concentration of CFU / mL exceeds a certain level.

[0025] High-temperature inactivation treatment: The fermentation broth in the fermenter is gradually heated to 80°C and kept at a constant temperature for 30 minutes. During this period, the broth is stirred at a low speed of 50 rpm to ensure that the broth is heated evenly. After inactivation, samples are aseptically taken, spread on MRS solid plates, and anaerobically incubated at 37°C for 48 hours. If no colonies grow on the plates, the inactivation is considered complete.

[0026] Concentration and freeze-drying: The completely inactivated fermentation broth was transferred to a tubular centrifuge and centrifuged at 8000 rpm for 15 min. The bacterial precipitate and fermentation supernatant were collected separately. The bacterial precipitate was resuspended with an equal volume of the original fermentation supernatant and thoroughly mixed to obtain a bacterial-metabolite suspension. The suspension was transferred to a freeze-drying tray, pre-frozen at -40℃ for 4 h, and then transferred to a vacuum freeze dryer. The freeze dryer was sublimated and dried at a cold trap temperature of -55℃ and a vacuum degree of 10 Pa for 24 h. After being removed, the liquid was pulverized and passed through a 100-mesh sieve to obtain the inactivated Lactobacillus plantarum prebiotic powder.

[0027] Tests showed that the inactivated probiotic powder contained ≥1×10¹¹ CFU / g of live bacteria (calculated based on the number of live bacteria before inactivation), and had a moisture content of ≤5%. The product contained intact bacterial fragments, peptidoglycan, lipoteichoic acid and other cell wall components, as well as all extracellular metabolites such as lactic acid, bacteriocins and short-chain fatty acids, and could be directly used in the formulation of toothpaste compositions.

[0028] Example 1 The composition of the composition is as follows, based on parts by weight: Targeted anti-inflammatory nanospheres: 0.8 parts Barrier repair complex: 0.3 parts (recombinant type III humanized collagen: oligomeric sodium hyaluronate = 1:2) Inactivated Lactobacillus plantarum biogener: 0.2 parts Toothpaste base ingredients: 30 parts hydrated silica, 15 parts glycerin, 15 parts sorbitol, 2 parts sodium lauroyl sarcosinate, 1 part potassium cocoyl glycinate, 0.8 parts sodium carboxymethyl cellulose, 0.2 parts xanthan gum, 0.2 parts sodium saccharin, 1 part edible peppermint flavor, 33.5 parts purified water. Preparation process (1) Preparation of targeted anti-inflammatory nanospheres ① Weigh 10g of hydroxypropyl-β-cyclodextrin, add it to 100mL of purified water, stir at 300rpm until completely dissolved, and prepare a cyclodextrin aqueous solution; ② Weigh 0.3g of resveratrol and 0.6g of EGCG, dissolve them in 20mL of anhydrous ethanol, and sonicate until completely clear to obtain an ethanol solution of the active ingredient; ③ Slowly add the active ingredient ethanol solution to the cyclodextrin aqueous solution. After the addition is complete, adjust the system temperature to 40℃ and stir at a constant temperature of 400rpm for 3 hours to incorporate the active ingredient. ④ After the inclusion is completed, the solution is spray-dried with an inlet air temperature of 120℃ and an outlet air temperature of 60℃. The dried nanosphere powder is then collected. ⑤ Weigh 5g of the above nanosphere powder, add it to 50mL of MES buffer solution with pH=6.0, disperse it evenly by sonication, add 0.15g of EDC and 0.1g of NHS, and activate at 25℃ for 30min; ⑥ Add 0.12g of ICAM-1 targeting peptide to the activated system, transfer to a 4℃ environment, and stir at low speed for 15h. ⑦ After the reaction is complete, the solution is placed in a dialysis bag (molecular weight cutoff 10kDa) and dialyzed with purified water for 24 hours to remove unreacted target peptides and activators; ⑧ The dialysis solution was freeze-dried to obtain targeted anti-inflammatory nanosphere powder. The average particle size was 210 nm, the total drug loading was 9.2%, the encapsulation efficiency was 87.5%, and the amount of targeted peptide grafting was 2.1%.

[0029] (2) Preparation of toothpaste composition ① Preparation of aqueous matrix: Weigh glycerol, sorbitol, sodium carboxymethyl cellulose, xanthan gum and sodium saccharin according to the formula, add them to purified water, and stir at 500 rpm until the thickener is completely swollen and there are no lumps; heat the system to 50℃, add hydrated silica, and disperse at high speed at 800 rpm for 15 min until the system is uniform and free of particles to obtain the aqueous matrix; ② Preparation of active premix: Weigh recombinant type III humanized collagen, oligomeric sodium hyaluronate, and inactivated Lactobacillus plantarum biogener according to the formula, add 5 parts of purified water at 4℃, and stir at 300rpm until completely dissolved and dispersed, then set aside. ③Ointment mixing: Cool the aqueous matrix to 28°C, add sodium lauroyl sarcosinate and potassium cocoyl glycinate, stir at 400 rpm for 10 min until completely dissolved; add targeted anti-inflammatory nanosphere powder, and continue stirring for 15 min until evenly dispersed; ④ Degassing and homogenization: Slowly add the active premixed liquid, controlling the addition time to 10 min. After the addition is complete, keep stirring and turn on vacuum degassing for 20 min with a vacuum degree of -0.08 MPa. Finally, add edible peppermint flavoring and homogenize at 600 rpm for 5 min to obtain the toothpaste composition of this embodiment.

[0030] Example 2 The composition of the composition is as follows, based on parts by weight: Targeted anti-inflammatory nanospheres: 2 parts Barrier Repair Complex: 1 part (Recombinant Type III Humanized Collagen: Oligomeric Sodium Hyaluronate = 1:2) Inactivated Lactobacillus plantarum post-biotic: 0.8 parts Toothpaste base ingredients: 30 parts hydrated silica, 15 parts glycerin, 15 parts sorbitol, 2 parts sodium lauroyl sarcosinate, 1 part potassium cocoyl glycinate, 0.8 parts sodium carboxymethyl cellulose, 0.2 parts xanthan gum, 0.2 parts sodium saccharin, 1 part edible peppermint flavor, 31 parts purified water. Preparation process (1) Preparation of targeted anti-inflammatory nanospheres The preparation process of the nanospheres in this embodiment is the same as that in Example 1. The active ingredient feed ratio was adjusted, and the final product had a total drug loading of 10.5%, an encapsulation efficiency of 89.2%, an average particle size of 225 nm, and a target peptide grafting amount of 2.3%.

[0031] (2) Preparation of toothpaste composition The preparation process is exactly the same as in Example 1, except that the amount of each component is adjusted according to the formula of this example.

[0032] Example 3

[0033] The composition of the composition is as follows, based on parts by weight: Targeted anti-inflammatory nanospheres: 4.5 parts Barrier repair complex: 2.5 parts (recombinant type III humanized collagen: oligomeric sodium hyaluronate = 1:2) Lactobacillus plantarum inactivated postbiotic: 1.8 parts Toothpaste base ingredients: 30 parts hydrated silica, 15 parts glycerin, 15 parts sorbitol, 2 parts sodium lauroyl sarcosinate, 1 part potassium cocoyl glycinate, 0.8 parts sodium carboxymethyl cellulose, 0.2 parts xanthan gum, 0.2 parts sodium saccharin, 1 part edible peppermint flavor, 26 parts purified water. Preparation process (1) Preparation of targeted anti-inflammatory nanospheres The preparation process was the same as in Example 1. The final product had a total drug loading of 11.2%, an encapsulation efficiency of 88.7%, an average particle size of 230 nm, and a target peptide grafting amount of 2.2%.

[0034] (2) Preparation of toothpaste composition The preparation process is exactly the same as in Example 1, except that the amount of each component is adjusted according to the formula of this example.

[0035] Comparative Example 1 The targeted anti-inflammatory nanospheres were removed, and free resveratrol and EGCG (0.07 parts resveratrol and 0.14 parts EGCG) were added in the same amount as those used in Example 2. Purified water was added by 1.79 parts to make up the total mass. The remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the effect of targeted encapsulated microspheres on enhancing the efficacy of the active ingredient.

[0036] Comparative Example 2 Hydroxypropyl-β-cyclodextrin nanospheres without ICAM-1 targeting peptide grafts were used, with the remaining components, amounts, and preparation process identical to those in Example 2. This comparative example was used to verify the contribution of the targeting peptide to the enrichment effect on inflammatory sites.

[0037] Comparative Example 3 The targeted nanospheres encapsulated only resveratrol, with the total drug loading consistent with Example 2. They did not contain EGCG, and the remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the synergistic effect of the combination of resveratrol and EGCG.

[0038] Comparative Example 4 The targeted nanospheres encapsulated only EGCG, with the total drug loading consistent with Example 2. They did not contain resveratrol, and the remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the synergistic effect of the combination of resveratrol and EGCG.

[0039] Comparative Example 5 The barrier repair complex was removed, and purified water was added by one part to make up the total mass. The remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the effect of the barrier repair system on long-term efficacy.

[0040] Comparative Example 6 The barrier repair component only added 0.33 parts of recombinant type III humanized collagen (consistent with the collagen content in Example 2), removed oligomeric sodium hyaluronate, and added 0.67 parts of purified water to make up the total mass. The remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the synergistic effect of the combination of collagen and oligomeric HA.

[0041] Comparative Example 7 The barrier repair component only added 0.67 parts of oligomeric sodium hyaluronate (consistent with the oligomeric HA content in Example 2), removed recombinant type III humanized collagen, and added 0.33 parts of purified water to make up the total mass. The remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the synergistic effect of the combination of collagen and oligomeric HA.

[0042] Comparative Example 8 After removing the inactivated Lactobacillus plantarum biotic, 0.8 parts of purified water were added to make up the total mass. The remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the effect of the microecological regulation unit on reducing the recurrence rate.

[0043] Comparative Example 9 Sodium lauroyl sarcosinate and potassium cocoyl glycinate were replaced with 3 parts sodium dodecyl sulfate (SLS), and the remaining components and preparation process were the same as in Example 2. This comparative example was used to verify the necessity of a mild excipient system.

[0044] Comparative Example 10 Simultaneously, the targeted anti-inflammatory nanospheres and barrier repair complex were removed, retaining only the inactivated Lactobacillus plantarum prebiotic and basic excipients. Purified water was added to bring the total to 100 parts, and the remaining preparation process was the same as in Example 2. This comparative example was used to verify the overall efficacy level after the core functional unit was missing.

[0045] Performance testing Detection methods 1. Rat gingivitis model and gingival bleeding index (GBI) detection One hundred and forty SPF-grade male SD rats, weighing 200±20g, were selected. A chronic gingivitis model was established by ligating the mandibular first molar with silk sutures and applying Porphyromonas gingivalis bacterial solution. The model was confirmed to be successful 7 days after modeling (GBI≥2). The rats were randomly divided into 15 groups of 10 rats each, corresponding to 3 examples, 10 comparative examples, and a blank model group.

[0046] Rats were treated twice daily with the corresponding toothpaste, 0.1g each time, while the control group received an equal volume of physiological saline. This intervention lasted for 14 consecutive days. Gingival bleeding index (GBI) was measured before intervention, on day 7, and on day 14: a standardized periodontal probe was used to gently probe the gingival sulcus, and bleeding was observed within 30 seconds. Scoring criteria were: 0 points = no bleeding; 1 point = punctate bleeding after probing; 2 points = linear bleeding after probing; 3 points = spontaneous bleeding. The mean GBI value for each group was calculated.

[0047] After 14 days of intervention, medication was stopped, and the animals were fed normally for 14 days. The GBI value was then measured again, and the bleeding recurrence rate was calculated: Recurrence rate = (GBI after medication discontinuation - GBI after 14 days of intervention) / GBI after 14 days of intervention × 100%.

[0048] 2. Anti-inflammatory and capillary permeability testing Fourteen days after intervention, six rats were randomly selected from each group, and they were sacrificed after blood was collected from the abdominal aorta. Gingival tissue was harvested from the mandibles. A 10% tissue homogenate was prepared, and the relative expression level of phosphorylated NF-κBp65, the content of IL-6 and TNF-α, and the activity of myeloperoxidase (MPO) in the gingival tissue were detected using an ELISA kit.

[0049] The Evans blue exudation method was used to detect gingival capillary permeability: 2% Evans blue saline solution (5 mL / kg) was injected into the tail vein 30 min before sacrifice. After sacrifice, gingival tissue was collected, weighed, and placed in formamide. It was soaked at 45℃ for 48 h, centrifuged, and the supernatant was collected. The absorbance was measured at 620 nm. The Evans blue exudate (μg / g tissue) was calculated according to the standard curve to reflect capillary permeability.

[0050] 3. Gingival barrier repair function test ① Human gingival epithelial cell (HGEC) barrier function: HGEC cells were cultured in Transwell chambers. After a dense monolayer was formed, 10% toothpaste extract was added and cultured for 24 hours. Transepithelial resistance (TEER) was measured using a cell resistance meter. The blank culture medium group was used as a control, and the relative TEER value was calculated.

[0051] ② Human gingival fibroblast (HGF) proliferation rate: HGF cells were seeded in 96-well plates, and cultured for 48 hours with toothpaste extract of different concentrations. Cell proliferation rate was detected by CCK-8 assay, with the blank control group having a cell proliferation rate of 100%.

[0052] 4. Antibacterial activity and microbial diversity detection ① Inhibition rate of Porphyromonas gingivalis: The Oxford cup agar diffusion method was used. Porphyromonas gingivalis bacterial suspension was evenly spread on blood agar plates, Oxford cups were placed and 200 μL of toothpaste extract was injected. After anaerobic culture for 48 h, the diameter of the inhibition zone was measured and the inhibition rate was calculated.

[0053] ②Inhibition rate of Streptococcus salivarius: The diameter of the inhibition zone against Streptococcus salivarius (beneficial bacteria in the oral cavity) was measured using the same method to evaluate the inhibitory selectivity.

[0054] ③ Periodontal microbiota diversity: 14 days after intervention, subgingival plaque samples were taken from rats, total bacterial DNA was extracted, and 16S rRNA gene V3-V4 region was sequenced to calculate the Shannon diversity index, which reflects the richness and evenness of the microbiota.

[0055] 5. Oral mucosal irritation test Seventy healthy rabbits were randomly divided into 14 groups of 5 rabbits each. 0.5g of toothpaste was applied to one cheek mucosa of each rabbit, while saline solution was applied to the other cheek as a control. This was done once daily for 7 consecutive days. The condition of the mucosa was observed daily and scored according to a rating scale: 0 points = no abnormality; 1 point = mild congestion; 2 points = significant congestion and edema; 3 points = erosion and ulceration. The average stimulation score was calculated: ≤0.5 for no irritation, 0.5-2 for mild irritation, and >2 for moderate or severe irritation.

[0056] 6. Accelerate stability testing The toothpaste samples from each embodiment and the comparative example were sealed and placed in a constant temperature and humidity chamber at 37°C and 75% relative humidity for accelerated storage for 3 months. The contents of resveratrol and EGCG after initial storage were detected by high performance liquid chromatography, and the retention rate of active ingredients was calculated as follows: Retention rate = Content after storage / Initial content × 100%.

[0057] Test results Table 1 Comparison of gingival bleeding index and recurrence rate among different groups Example 1 2.31±0.22 1.25±0.18 0.62±0.11 18.3 Example 2 2.29±0.24 0.87±0.13 0.31±0.08 8.7 Example 3 2.33±0.21 0.79±0.12 0.27±0.07 7.9 Comparative Example 1 2.30±0.23 1.72±0.21 1.24±0.16 35.6 Comparative Example 2 2.28±0.25 1.34±0.19 0.85±0.14 22.4 Comparative Example 3 2.32±0.22 1.41±0.20 0.93±0.15 25.1 Comparative Example 4 2.27±0.24 1.53±0.21 1.02±0.16 27.3 Comparative Example 5 2.31±0.23 0.95±0.15 0.48±0.10 41.2 Comparative Example 6 2.29±0.22 1.02±0.16 0.57±0.11 32.8 Comparative Example 7 2.30±0.24 1.10±0.17 0.65±0.12 30.5 Comparative Example 8 2.28±0.23 0.92±0.14 0.38±0.09 31.7 Comparative Example 9 2.32±0.21 1.21±0.18 0.76±0.13 38.9 Comparative Example 10 2.31±0.23 1.95±0.24 1.68±0.20 47.2 Model blank group 2.30±0.22 2.24±0.23 2.17±0.21 - Table 2 Comparison of anti-inflammatory and capillary permeability indices among groups Example 1 0.42±0.05 86.3±7.2 52.7±5.4 12.5±1.3 Example 2 0.21±0.03 45.2±4.6 28.1±3.2 6.8±0.7 Example 3 0.19±0.03 41.8±4.3 25.6±2.9 6.2±0.6 Comparative Example 1 0.68±0.07 132.5±10.8 84.3±7.6 19.7±1.8 Comparative Example 2 0.48±0.06 95.7±8.1 59.4±5.8 14.2±1.5 Comparative Example 3 0.53±0.06 104.2±9.3 65.8±6.3 15.6±1.6 Comparative Example 4 0.57±0.07 112.6±9.8 71.2±6.7 16.8±1.7 Comparative Example 5 0.25±0.03 52.4±5.1 32.7±3.5 7.5±0.8 Comparative Example 6 0.30±0.04 61.8±5.7 38.5±4.1 8.9±0.9 Comparative Example 7 0.33±0.04 68.3±6.2 42.1±4.4 9.7±1.0 Comparative Example 8 0.24±0.03 49.6±4.8 30.4±3.3 7.2±0.7 Comparative Example 9 0.41±0.05 78.5±6.9 48.2±4.9 11.8±1.2 Comparative Example 10 0.82±0.08 156.7±12.4 102.5±8.9 23.4±2.1 Model blank group 1.00±0.09 189.3±14.2 124.6±10.3 28.7±2.5 Table 3 Comparison of gingival barrier repair function among groups Example 1 128.5±6.2 121.3±5.7 Example 2 152.7±7.5 146.8±6.9 Example 3 157.2±7.8 151.4±7.2 Comparative Example 1 105.2±4.8 103.7±4.5 Comparative Example 2 118.6±5.6 112.4±5.1 Comparative Example 3 112.4±5.3 108.9±4.9 Comparative Example 4 110.8±5.1 107.2±4.8 Comparative Example 5 103.5±4.7 102.1±4.4 Comparative Example 6 122.3±5.9 125.6±6.0 Comparative Example 7 115.7±5.5 113.8±5.4 Comparative Example 8 148.4±7.2 142.5±6.7 Comparative Example 9 82.6±3.9 89.3±4.2 Comparative Example 10 101.2±4.6 100.8±4.6 Blank control group 100.0±4.5 100.0±4.6 Table 4 Comparison of antibacterial effects and microbial diversity among different groups Example 1 18.2±1.1 0 3.82±0.15 Example 2 22.5±1.3 0 4.15±0.18 Example 3 23.7±1.4 0 4.21±0.19 Comparative Example 1 12.4±0.8 0 3.41±0.13 Comparative Example 2 16.3±1.0 0 3.67±0.14 Comparative Example 3 14.7±0.9 0 3.52±0.13 Comparative Example 4 17.1±1.0 0 3.59±0.14 Comparative Example 5 21.8±1.2 0 4.08±0.17 Comparative Example 6 22.1±1.3 0 4.10±0.17 Comparative Example 7 22.3±1.2 0 4.12±0.18 Comparative Example 8 22.0±1.2 0 3.26±0.12 Comparative Example 9 20.6±1.1 0 3.74±0.15 Comparative Example 10 7.5±0.5 0 3.35±0.13 Model blank group 0 0 2.94±0.11 Table 5 Comparison of mucosal irritation and stability of active ingredients in each group Example 1 0.21±0.05 Non-irritating 92.3 90.7 Example 2 0.18±0.04 Non-irritating 93.5 91.9 Example 3 0.20±0.05 Non-irritating 92.8 91.2 Comparative Example 1 0.23±0.05 Non-irritating 61.4 58.2 Comparative Example 2 0.19±0.04 Non-irritating 93.1 91.5 Comparative Example 3 0.20±0.05 Non-irritating 92.7 - Comparative Example 4 0.19±0.04 Non-irritating - 91.1 Comparative Example 5 0.22±0.05 Non-irritating 93.2 91.7 Comparative Example 6 0.21±0.05 Non-irritating 93.0 91.4 Comparative Example 7 0.20±0.05 Non-irritating 92.9 91.3 Comparative Example 8 0.19±0.04 Non-irritating 93.3 91.6 Comparative Example 9 1.87±0.21 Mild stimulation 84.6 80.3 Comparative Example 10 0.22±0.05 Non-irritating - - The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A toothpaste composition for improving bleeding gums, characterized in that, By weight, it consists of the following components: 0.5-5 parts targeted anti-inflammatory nanospheres, 0.2-3 parts barrier repair complex, 0.1-2 parts inactivated Lactobacillus plantarum biotic, and 85-98 parts toothpaste base excipients.

2. The toothpaste composition for improving gingival bleeding according to claim 1, characterized in that, The targeted anti-inflammatory nanospheres are hydroxypropyl-β-cyclodextrin nanospheres with ICAM-1 targeting peptides covalently grafted on their surface. The nanospheres contain a complex of resveratrol and epigallocatechin gallate in a mass ratio of 1:1 to 1:

3. The barrier repair compound is composed of recombinant type III humanized collagen and oligomeric sodium hyaluronate in a mass ratio of 1:1 to 1:

5.

3. The toothpaste composition for improving gingival bleeding according to claim 1, characterized in that, In the targeted anti-inflammatory nanospheres, the total drug loading of resveratrol and epigallocatechin gallate is 8%-15%, and the encapsulation efficiency is ≥85%; the grafting amount of the ICAM-1 targeting peptide is 1.5%-3% of the mass of the microspheres.

4. The toothpaste composition for improving gingival bleeding according to claim 1, characterized in that, The average particle size of the targeted anti-inflammatory nanospheres is 150-300 nm; the molar substitution degree of the hydroxypropyl-β-cyclodextrin is 4-7.

5. The toothpaste composition for improving gingival bleeding according to claim 1, characterized in that, The inactivated Lactobacillus plantarum biogener is prepared by fermentation culture, high-temperature inactivation, and freeze-drying of Lactobacillus plantarum, wherein the bacterial cell content is ≥1×10¹¹CFU / g, and includes intact bacterial cell fragments and extracellular metabolites.

6. The toothpaste composition for improving gingival bleeding according to claim 1, characterized in that, By weight, the toothpaste base excipients consist of the following components: 20-40 parts abrasive, 20-35 parts humectant, 1-5 parts amino acid surfactant, 0.5-2 parts thickener, 0.1-0.5 parts sweetener, 0.5-1.5 parts edible flavoring, and purified water to make up to 100 parts.

7. The toothpaste composition for improving gingival bleeding according to claim 6, characterized in that, The friction agent is one or both of hydrated silica and aluminum hydroxide; The moisturizer is at least two of glycerin, sorbitol, and polyethylene glycol-400; The surfactant is one or two of sodium lauroyl sarcosinate and potassium cocoyl glycinate. The thickener is at least two of sodium carboxymethyl cellulose, xanthan gum, and carbomer.

8. A method for preparing a toothpaste composition for improving gingival bleeding as described in any one of claims 1-7, characterized in that, Includes the following steps: S1: Hydroxypropyl-β-cyclodextrin was dissolved in purified water, and an ethanol solution of resveratrol and epigallocatechin gallate was added. After constant temperature stirring and encapsulation, the mixture was spray-dried to obtain nanosphere powder. The nanosphere powder was activated by EDC / NHS, and ICAM-1 targeting peptide was added for covalent grafting. After dialysis purification and freeze-drying, targeted anti-inflammatory nanospheres were obtained. S2: Add humectant, thickener and sweetener to purified water, stir until completely swollen, heat to 45-55℃ and then add abrasive, disperse at high speed until uniform and free of particles to obtain aqueous matrix; S3: Add the barrier repair complex and inactivated Lactobacillus plantarum biogen to purified water at 4°C and stir at low speed until completely dissolved and dispersed to obtain an active premix; S4: Cool the aqueous matrix to 25-30℃, add the surfactant and stir at low speed until completely dissolved, then add the targeted anti-inflammatory nanospheres and disperse evenly; then slowly add the active premixed liquid, and after the addition is complete, vacuum degas for 15-30 minutes, add the edible flavoring and continue to homogenize and stir to obtain the toothpaste composition for improving gingival bleeding.

9. The preparation method according to claim 8, characterized in that, In step S1, the inclusion reaction temperature is 35-45℃, the stirring rate is 300-500rpm, and the inclusion time is 2-4h; the pH of the targeted peptide grafting reaction system is 5.5-6.5, the reaction temperature is 2-8℃, and the reaction time is 12-18h.

10. The use of the toothpaste composition for improving gingival bleeding as described in any one of claims 1-7 in the preparation of oral care products that improve gingival bleeding, repair the gingival epithelial barrier, and regulate the periodontal microecology.