Application of recombinant human glaze matrix protein in anti-periodontitis oral care product
By combining recombinant human enamel matrix protein with stabilizers and promoters, an anti-periodontalgia oral care product was prepared, which solved the problem of periodontitis treatment in existing technologies and achieved multiple functional effects such as high-efficiency penetration, safe antibacterial properties, and promotion of tissue repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU XUELIANG BIOTECHNOLOGY DEVELOPING CO LTD
- Filing Date
- 2026-01-21
- Publication Date
- 2026-05-26
AI Technical Summary
Existing treatments and care methods for periodontitis are insufficient to completely remove pathogenic biofilms within deep periodontal pockets. Long-term use of antibiotics can easily lead to drug resistance and microecological imbalance. Common oral care products are prone to inactivation in complex environments and lack multiple functions, making them unable to effectively inhibit bacterial recolonization and promote tissue repair.
Recombinant human ameliorative matrix protein is combined with poloxamer 188, sodium lauroyl sarcosinate, and other ingredients to form a stable system. Sodium hyaluronate, tourmaline powder, and liposome propyl gallate are added to prepare a mouthwash or toothpaste. Through synergistic anti-inflammatory effects, it promotes the migration and attachment of gingival fibroblasts and enhances the antioxidant damage effect.
It achieves high solubility and bioactivity in the oral environment, significantly reduces gingival index and inflammatory factor levels in periodontitis model animals, promotes gingival soft tissue healing and improves local microcirculation, and provides multifunctional adjunctive treatment for periodontitis.
Smart Images
Figure SMS_2 
Figure SMS_3 
Figure SMS_4
Abstract
Description
Technical Field
[0001] This invention relates to the application of recombinant human enamel matrix protein in oral care products for periodontitis, and belongs to the field of special protein application technology. Background Technology
[0002] Current adjunctive treatments and daily care methods for periodontitis have significant limitations and shortcomings: While mechanical debridement as a basic treatment can remove plaque and tartar, it is difficult to completely remove pathogenic biofilms in deep periodontal pockets, and it cannot effectively inhibit bacterial recombination or regulate abnormal host immune inflammatory responses, leading to a high recurrence rate; although topical or systemic antibiotics can suppress pathogens in the short term, long-term use can easily induce bacterial resistance, disrupt the oral microecological balance, and cause side effects such as gastrointestinal discomfort; commonly available antibacterial mouthwashes mainly rely on broad-spectrum bactericidal agents such as chlorhexidine and cetylpyridinium chloride. While these ingredients can reduce plaque to some extent, long-term use may lead to adverse reactions such as tooth discoloration and altered taste. Furthermore, their mechanism of action is singular, focusing on sterilization while lacking the ability to promote periodontal tissue regeneration, and offering almost no repair effect on existing periodontal soft tissue damage and alveolar bone resorption. In addition, some high-end oral care products attempting to incorporate bioactive factors face bottlenecks such as the easy inactivation of active ingredients in the complex oral environment, short residence time at the site of action, difficulty in penetrating biomembrane barriers, high production costs, and poor formula stability, limiting their widespread application and actual therapeutic effects. Therefore, there is an urgent need in this field for an oral care solution that can penetrate efficiently, safely kill bacteria, and simultaneously possess multiple functions such as anti-inflammatory and periodontal tissue repair promotion, while also maintaining stable performance. Summary of the Invention
[0003] To address the shortcomings of existing technologies, this invention provides the application of recombinant human enamel matrix protein in the preparation of an anti-periodontal oral care product, wherein the anti-periodontal oral care product comprises the following components:
[0004] Recombinant human ameliorative matrix protein 0.001-0.1 wt%; glycerol 1-5 wt%; sorbitol 5-10 wt%; poloxamer 188 0.1-0.5 wt%; sodium lauroyl sarcosinate 0.1-0.5 wt%; potassium nitrate 0.1-1 wt%; sucralose 0.001-0.01 wt%; xylitol 0.1-1 wt%; methylparaben 0.1-0.3 wt%; propylene glycol 0.1-1.5 wt%; PEG-40 hydrogenated castor oil 0.1-1 wt%; domiphen 0.005-0.2 wt%; balance: purified water and fragrance.
[0005] Preferably, the mass ratio of poloxamer 188 to sodium lauroyl sarcosinate is 1:1 to 1:2.
[0006] Preferably, the pH value of the anti-periodontalgia oral care product is adjusted to 6.5-7.5.
[0007] Preferably, the anti-periodontic oral care product is mouthwash, oral spray, or toothpaste.
[0008] Preferably, the anti-periodontalgia oral care product further contains 0.5-2 wt% sodium hyaluronate.
[0009] Preferably, the molecular weight of the sodium hyaluronate is 800,000 to 1,500,000 Daltons.
[0010] Preferably, the anti-periodontic oral care product further comprises 0.5-2 wt% of submicron-sized tourmaline powder, wherein the tourmaline powder has a particle size of 200-500 nm and is composed of magnesium iron lithium tourmaline.
[0011] Preferably, the anti-periodontic oral care product further comprises 0.1-0.5 wt% of liposome-encapsulated propyl gallate.
[0012] Preferably, the method for preparing the liposome-encapsulated propyl gallate includes: dissolving lecithin and cholesterol in anhydrous ethanol at a mass ratio of 3:1, rotary evaporating to form a lipid film, adding PBS solution (pH 7.4) containing 0.5% propyl gallate, hydrating at 55°C for 30 min, and extruding through a 0.22 μm filter membrane.
[0013] Preferably, when the product is in the form of mouthwash, the method for preparing the anti-periodontic oral care product described above includes the following steps:
[0014] (1) Dissolve glycerol, sorbitol, xylitol, propylene glycol, potassium nitrate, sucralose, and domiphen in most of the purified water and stir until completely dissolved to obtain an aqueous phase;
[0015] (2) Dissolve poloxamer 188 and sodium lauroyl sarcosinate in the remaining purified water, or mix with PEG-40 hydrogenated castor oil and methylparaben and add to the aqueous phase, stirring until completely dissolved or evenly dispersed;
[0016] (3) Add the aqueous solution of recombinant human ameliorative matrix protein and stir to mix well;
[0017] (4) Adjust the pH of the system to 6.5-7.5 using citrate-sodium citrate buffer solution;
[0018] (5) Add flavoring, add purified water to the full volume, stir well and then fill.
[0019] Preferably, sodium hyaluronate is added after step (4) and before step (5), and stirred until fully swollen.
[0020] Preferably, after step (4) and before step (5), submicron-sized tourmaline powder is added and homogeneously dispersed.
[0021] Preferably, after step (4) and before step (5), liposome-encapsulated propyl gallate is added and slowly stirred to mix.
[0022] Preferably, when the product is in the form of a lotion-type mouthwash, the method for preparing the anti-periodontic oral care product used above includes the following steps:
[0023] (1) Dissolve glycerol, sorbitol, xylitol, propylene glycol, potassium nitrate, sucralose, and domiphen in a portion of purified water and stir until completely dissolved to obtain aqueous phase A;
[0024] (2) Poloxamer 188, sodium lauroyl sarcosinate, PEG-40 hydrogenated castor oil, and methylparaben were mixed and dissolved under heating conditions (60-70℃) to obtain oil phase B;
[0025] (3) Under high-speed shearing (8000-10000 rpm), oil phase B is slowly added to aqueous phase A and sheared for 5-10 minutes to obtain primary emulsion;
[0026] (4) Cool the colostrum to below 40°C, add an aqueous solution of recombinant human ameliorative matrix protein, and stir slowly to mix well;
[0027] (5) Adjust the pH of the system to 6.5-7.5 using citrate-sodium citrate buffer solution;
[0028] (6) Add flavoring, add purified water to the full volume, homogenize and then fill.
[0029] Preferably, the method for preparing the recombinant human ameliorative matrix protein includes:
[0030] a. The optimized human ameliorative matrix protein DNA fragment was ligated into the pET-32a(+) vector and transformed into BL21(DE3) competent cells;
[0031] b. After IPTG induction, the bacterial cells were resuspended in a buffer containing 20 mM Tris, 200 mM NaCl, and 5 mM imidazole (pH 8.0) and homogenized at 1000 bar for lysis.
[0032] c. The lysis buffer and NI-NTA affinity chromatography medium are mixed at a volume ratio of 5:1, and then washed with 50-100 mM imidazole and eluted with 200-300 mM imidazole.
[0033] d. Add TEV enzyme (1 KU / g protein) to the elution buffer and digest at 30°C for 2 h;
[0034] e. The enzyme digestion product was precipitated with 56 g / L ammonium sulfate, and the precipitate was collected by centrifugation;
[0035] f. Freeze-dry the precipitate to obtain the final product.
[0036] Preferably, the optimized amino acid sequence of the human ameliorative matrix protein in step a is as follows: .
[0037] Preferably, in step c, the concentration of eluting imidazole is 75 mM and the concentration of eluting imidazole is 250 mM; the TEV enzyme used in step d is prepared by an E. coli expression system with a specific activity ≥8000 U / mg; the purity of the final product is ≥90% as determined by SDS-PAGE, and the endotoxin content is <0.1 EU / mg.
[0038] The beneficial effects of this invention are:
[0039] This invention successfully integrates recombinant human ameliorative matrix proteins into an oral care matrix containing wetting agents, stabilizers, desensitizing agents, and preservatives, pioneering a new application for them in the field of anti-periodontitis. Through a composite stabilization system composed of poloxamer 188 and sodium lauroyl sarcosinate, the key challenge of rhEMPs' easy inactivation in the physiological pH oral environment is effectively solved, ensuring that it maintains a solubility and bioactivity of over 98% throughout the product's shelf life. The rhEMPs in this basic formula exhibit synergistic anti-inflammatory and antibacterial effects with potassium nitrate and domiphen, and can effectively promote the migration and attachment of gingival fibroblasts. Further introduction of advanced components such as sodium hyaluronate, tourmaline powder, and liposomal propyl gallate reveals unexpected synergistic benefits in promoting gingival soft tissue healing, improving local microcirculation, and enhancing antioxidant damage resistance. Animal experiments show that it can significantly reduce the gingival index, probing depth, and levels of inflammatory factors IL-1β and TNF-α in periodontitis model animals, thus achieving effective adjunctive treatment for periodontitis. Detailed Implementation
[0040] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.
[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.
[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.
[0043] Example:
[0044] I. Preparation of Recombinant Human Ameliorative Matrix Proteins (rhEMPs)
[0045] 1. Preparation of experimental materials: Reagents: pET-32a(+) plasmid, BL21(DE3) competent cells, ampicillin, IPTG (isopropyl-β-D-thiogalactoside), Tris, NaCl, imidazole, ammonium sulfate (all analytical grade); Equipment: clean bench, constant temperature shaker, high pressure homogenizer, high speed homogenizer, 150 mesh sieve, tubular centrifuge, freeze dryer, SDS-PAGE electrophoresis apparatus.
[0046] 2. Construction of recombinant plasmids
[0047] Sequence optimization: The signal peptide of the full-length human ameliorative matrix protein was removed, and the optimized amino acid sequence was retained.
[0048] After codon optimization, the DNA sequence was obtained:
[0049] The optimized DNA fragment was ligated to the pET-32a(+) plasmid to construct a recombinant plasmid.
[0050] 3. Culture of recombinant strains
[0051] Plasmid transformation: Remove BL21(DE3) competent cells from -80℃, thaw them in an ice bath for 5 minutes, add the recombinant plasmid, mix gently, and incubate in an ice bath for 25 minutes; heat shock in a 42℃ water bath for 45 seconds, then immediately return to the ice bath and incubate for 2 minutes; add 700 μl of antibiotic-free LB medium, and revive in a shaker at 37℃ and 200 rpm for 60 minutes; centrifuge at 5000 rpm for 1 minute, resuspend the bacterial block in 100 μl of supernatant, spread it on an LB agar plate containing ampicillin, and incubate overnight in an inverted 37℃ incubator.
[0052] Single colony screening and preservation: Pick a single colony and inoculate it into 20 ml of LB liquid medium containing ampicillin. Shake overnight at 37°C and 200 rpm for 12 hours. Take 10 ml of bacterial culture and mix it with 10 ml of 50% sterile glycerol. After aliquoting, store at -80°C.
[0053] 4. Induced expression
[0054] Expanded culture: Inoculate the preservative solution into a 500ml Erlenmeyer flask (containing 50ml of LB medium with ampicillin) at a ratio of 1%, and shake at 37℃ and 180rpm until the OD600 value is 0.6.
[0055] Induction of expression: Add IPTG to a final concentration of 0.4 mM, shake at 34°C and 120 rpm for 3 hours; after 10 minutes on ice, centrifuge at 4°C and 5000×g for 10 minutes, discard the supernatant, and store the bacterial pellet at -20°C.
[0056] 5. Protein purification
[0057] Lysis: Take the bacterial cell precipitate, add 9 times the weight of Ni equilibration solution (20 mM Tris + 200 mM NaCl + 5 mM imidazole, pH 8.0), and resuspend in a high-speed homogenizer; homogenize twice in a high-pressure homogenizer at 1000 bar and 5 °C to obtain the lysate.
[0058] Ni column affinity chromatography: Chuan BioNINTARP medium was mixed with Ni equilibration buffer at a volume ratio of 1:5, stirred and incubated for 30 minutes, the medium was retained through a 150-mesh sieve and the solution was drained; the lysis buffer was mixed with the medium at a volume ratio of 5:1, stirred and incubated for 30 minutes, the medium was retained through a sieve; the medium was washed with Ni equilibration buffer at a volume ratio of 5:1 for 30 minutes, the medium was retained through a sieve; impurities were washed with Ni washing buffer containing 75 mM imidazole at a volume ratio of 5:1 for 30 minutes, the impurities were retained through a sieve; elution was carried out with Ni elution buffer containing 250 mM imidazole at a volume ratio of 2:1 for 30 minutes, and the eluent (preliminarily purified rhEMPs solution) was collected.
[0059] TEV digestion: Add self-produced TEV enzyme (specific activity ≥8000U / mg) at a ratio of 1KU / g protein, and digest in a 30℃ water bath for 2 hours.
[0060] Salting out: Add ammonium sulfate slowly at 56 g / L to the enzyme-digested solution while stirring. After dissolving, let stand for 1 hour. Centrifuge at 14000 rpm, feed flow rate of 1 L / min, and cooling water temperature of 5℃ to collect the precipitate. Rinse the precipitate with 56 g / L ammonium sulfate solution and centrifuge again to collect the precipitate.
[0061] Freeze-drying: The precipitate was placed in a freeze dryer and freeze-dried to obtain rhEMPs dry powder (purity ≥96% as determined by SDS-PAGE).
[0062] II. Raw Material Preparation
[0063] 1. Preparation of propyl gallate encapsulated in liposomes
[0064] Weigh 3g of lecithin and 1g of cholesterol, dissolve them in 50ml of anhydrous ethanol, and pour the solution into a round-bottom flask of a rotary evaporator; remove the anhydrous ethanol by rotary evaporation at 37℃ to form a uniform lipid film; add 50ml of PBS solution (pH 7.4) containing 0.5% propyl gallate; hydrate in a water bath at 55℃ for 30 minutes, stirring magnetically during the process to completely detach the lipid film; squeeze the mixture through a 0.22μm filter membrane to obtain a liposome-encapsulated propyl gallate suspension, and store at 4℃.
[0065] 2. Preparation of submicron-sized tourmaline powder
[0066] Magnesium iron lithium tourmaline powder with a particle size of 300nm (chemical formula: Na(Mg,Fe,Li)3Al6(BO3)3Si6O) was selected. 18 (OH)4) is sterilized at 121℃ for 30 minutes before use and then dried for later use.
[0067] Example 1
[0068] Formula (based on 100g):
[0069] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, Glycerin (Sinopharm): 4g, Sorbitol (Roquette): 7g, Poloxamer 188 (BASF, Pluronic® F-68): 0.3g, Sodium lauroyl sarcosinate (Sigma-Aldrich): 0.2g, Potassium nitrate (Sinopharm): 0.5g, Sucralose (Tyler): 0.005g, Xylitol (Roquette): 0.5g, Methylparaben (Clariant): 0.2g, Propylene glycol (Dow Chemical): 1.0g, PEG-40 hydrogenated castor oil (BASF, Cremophor® RH40): 0.5g, Domiphen (Shanghai Aladdin): 0.01g, Peppermint flavor (Givaudan): 0.05g, Purified water: make up to 100g.
[0070] Preparation method:
[0071] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0072] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0073] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0074] Adjusting the pH value: After rinsing the electrode of the precision pH meter with purified water, immerse it in the solution from step 3 and wait for the reading to stabilize. Use 0.1M citric acid solution to slowly add dropwise and 0.1M sodium citrate solution to assist in fine-tuning, precisely adjusting the pH value of the system to 7.0.
[0075] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0076] Example 2
[0077] Formula (based on 100g):
[0078] Recombinant human ameliorative matrix proteins (rhEMPs): 0.05g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.4g, sodium lauroyl sarcosinate: 0.4g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0079] Preparation method:
[0080] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in glycerol, sorbitol, xylitol, propylene glycol, potassium nitrate, sucralose, and domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0081] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in poloxamer 188, sodium lauroyl sarcosinate, PEG-40 hydrogenated castor oil, and methylparaben. Add 10g of purified water to this beaker. Place this small beaker in a 60℃ constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0082] Adding active protein: Take a 15ml centrifuge tube and accurately weigh out the rhEMPs powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0083] Adjusting the pH value: After rinsing the electrode of the precision pH meter with purified water, immerse it in the solution from step 3 and wait for the reading to stabilize. Use 0.1M citric acid solution to slowly add dropwise and 0.1M sodium citrate solution to assist in fine-tuning, precisely adjusting the pH value of the system to 7.2.
[0084] Volume adjustment and filling: Add peppermint flavoring to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0085] Example 3
[0086] Formula (based on 100g):
[0087] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.15g, sodium lauroyl sarcosinate: 0.1g, potassium nitrate: 0.2g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0088] Preparation method:
[0089] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in glycerol, sorbitol, xylitol, propylene glycol, potassium nitrate, sucralose, and domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0090] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in poloxamer 188, sodium lauroyl sarcosinate, PEG-40 hydrogenated castor oil, and methylparaben. Add 10g of purified water to this beaker. Place this small beaker in a 60℃ constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0091] Adding active protein: Take a 15ml centrifuge tube and accurately weigh out the rhEMPs powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0092] Adjusting the pH value: After rinsing the electrode of the precision pH meter with purified water, immerse it in the solution from step 3 and wait for the reading to stabilize. Use 0.1M citric acid solution to slowly add dropwise and 0.1M sodium citrate solution to assist in fine-tuning, precisely adjusting the pH value of the system to 7.0.
[0093] Volume adjustment and filling: Add peppermint flavoring to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g. Pour the final product into a 100ml plastic mouthwash bottle, tighten the cap, and label it.
[0094] Example 4
[0095] Formula (based on 100g):
[0096] Recombinant human ameliorative matrix protein (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, sodium hyaluronate: 1.5g, submicron tourmaline powder: 1.0g, liposome-encapsulated propyl gallate: 0.3g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0097] Preparation method:
[0098] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 70g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0099] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0100] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0101] Add sodium hyaluronate: While stirring slowly at 300 rpm, weigh 1.5g of sodium hyaluronate powder using an electronic balance. Very slowly and gradually sprinkle it onto the surface of the solution with a spatula to avoid clumping. After all the powder has been added, increase the stirring speed to 500 rpm and continue stirring for 2 hours, until the sodium hyaluronate is completely swollen and the solution becomes viscous.
[0102] Add tourmaline powder: Weigh 1.0g of tourmaline powder. Start the high-speed shear disperser IKA T25 at 10,000 rpm and slowly add the tourmaline powder. After all the powder has been added, continue high-speed shearing for 5 minutes to ensure the powder is evenly dispersed.
[0103] Adding liposomes: Stop shearing and resume slow stirring at 300 rpm. Weigh out the equivalent of a liposome suspension containing 0.3 g of propyl gallate and slowly add it dropwise to the system using a dropper. After the addition is complete, continue stirring slowly for 30 minutes.
[0104] Adjusting the pH value: After rinsing the electrode of the precision pH meter with purified water, immerse it in the solution from step 6 and wait for the reading to stabilize. Use 0.1M citric acid solution to slowly add dropwise and 0.1M sodium citrate solution to assist in fine-tuning, precisely adjusting the pH value of the system to 7.0.
[0105] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution, stabilizer solution, and liposome suspension with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0106] Comparative Example 1 (No stable system)
[0107] Formula (based on 100g):
[0108] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0109] Preparation method:
[0110] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0111] To dissolve the oily components: Take a 50ml beaker, add 0.2g of methylparaben to 1.0g of propylene glycol, heat slightly and stir to dissolve. Then add this solution along with 0.5g of PEG-40 hydrogenated castor oil to the large beaker from step 1, and stir gently to mix.
[0112] Adding active protein: Accurately weigh 0.01g of rhEMPs dry powder into a 15ml centrifuge tube. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution using a dropper or pipette. You will immediately observe the solution becoming cloudy and a large amount of white flocculent precipitate appearing.
[0113] Subsequent steps could not be performed, and the experiment was terminated.
[0114] Comparative Example 2 (Single Stabilizer - Poloxamer Only)
[0115] Formula (based on 100g):
[0116] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.5g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0117] Preparation method:
[0118] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0119] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.5g poloxamer 188, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0120] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0121] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0122] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0123] Comparative Example 3 (Single stabilizer - sodium lauroyl sarcosinate only)
[0124] Formula (based on 100g):
[0125] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, sodium lauroyl sarcosinate: 0.5g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0126] Preparation method:
[0127] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0128] Dissolving the stabilizer and oily components: Take a 100ml beaker and accurately weigh in 0.5g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0129] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0130] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0131] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0132] Comparative Example 4 (Imbalanced Stabilizer Ratio)
[0133] Formula (based on 100g):
[0134] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.1g, sodium lauroyl sarcosinate: 0.4g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0135] Preparation method:
[0136] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0137] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.1g poloxamer 188, 0.4g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0138] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0139] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0140] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0141] Comparative Example 5 (Basic + Hyaluronic Acid Only)
[0142] Formula (based on 100g):
[0143] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, sodium hyaluronate: 1.5g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0144] Preparation method:
[0145] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 70g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0146] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0147] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0148] Add sodium hyaluronate: While stirring slowly at 300 rpm, weigh 1.5g of sodium hyaluronate powder using an electronic balance. Very slowly and gradually sprinkle it onto the surface of the solution with a spatula to avoid clumping. After all the powder has been added, increase the stirring speed to 500 rpm and continue stirring for 2 hours, until the sodium hyaluronate is completely swollen and the solution becomes viscous.
[0149] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0150] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0151] Comparative Example 6 (Basic + Tourmaline Only)
[0152] Formula (based on 100g):
[0153] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, submicron tourmaline powder: 1.0g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0154] Preparation method:
[0155] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0156] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0157] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0158] Add tourmaline powder: Weigh 1.0g of tourmaline powder. Start the KA T25 high-speed shear disperser at 10,000 rpm and slowly add the tourmaline powder. After all the powder has been added, continue high-speed shearing for 5 minutes to ensure the powder is evenly dispersed.
[0159] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0160] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0161] Comparative Example 7 (Basic + Liposomes Only)
[0162] Formula (based on 100g):
[0163] Recombinant human ameliorative matrix protein (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, liposome-encapsulated propyl gallate (based on propyl gallate content): 0.3g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0164] Preparation method:
[0165] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0166] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0167] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0168] Add liposomes: Maintain slow stirring at 300 rpm. Weigh out the equivalent of a liposome suspension containing 0.3 g of propyl gallate and slowly add it dropwise into the system using a dropper. After the addition is complete, continue stirring slowly for 30 minutes.
[0169] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0170] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution, stabilizer solution, and liposome suspension with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0171] Comparative Example 8 (Basic + Hyaluronic Acid + Tourmaline)
[0172] Formula (based on 100g):
[0173] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, sodium hyaluronate: 1.5g, submicron tourmaline powder: 1.0g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0174] Preparation method:
[0175] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 70g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0176] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0177] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0178] Add sodium hyaluronate: While stirring slowly at 300 rpm, weigh 1.5g of sodium hyaluronate powder using an electronic balance. Very slowly and gradually sprinkle it onto the surface of the solution with a spatula to avoid clumping. After all the powder has been added, increase the stirring speed to 500 rpm and continue stirring for 2 hours, until the sodium hyaluronate is completely swollen and the solution becomes viscous.
[0179] Add tourmaline powder: Weigh 1.0g of tourmaline powder. Start the high-speed shear disperser IKA T25 at 10,000 rpm and slowly add the tourmaline powder. After all the powder has been added, continue high-speed shearing for 5 minutes to ensure the powder is evenly dispersed.
[0180] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0181] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0182] Comparative Example 9 (Basic + Hyaluronic Acid + Liposomes)
[0183] Formula (based on 100g):
[0184] Recombinant human ameliorative matrix protein (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, sodium hyaluronate: 1.5g, liposome-encapsulated propyl gallate: 0.3g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0185] Preparation method:
[0186] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 70g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0187] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0188] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0189] Add sodium hyaluronate: While stirring slowly at 300 rpm, weigh 1.5g of sodium hyaluronate powder using an electronic balance. Very slowly and gradually sprinkle it onto the surface of the solution with a spatula to avoid clumping. After all the powder has been added, increase the stirring speed to 500 rpm and continue stirring for 2 hours, until the sodium hyaluronate is completely swollen and the solution becomes viscous.
[0190] Add liposomes: Maintain slow stirring at 300 rpm. Weigh out the equivalent of a liposome suspension containing 0.3 g of propyl gallate and slowly add it dropwise into the system using a dropper. After the addition is complete, continue stirring slowly for 30 minutes.
[0191] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0192] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution, stabilizer solution, and liposome suspension with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0193] Comparative Example 10 (Basic + Tourmaline + Liposomes)
[0194] Formula (based on 100g):
[0195] Recombinant human ameliorative matrix protein (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, potassium nitrate: 0.5g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, submicron tourmaline powder: 1.0g, liposome-encapsulated propyl gallate (based on propyl gallate content): 0.3g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0196] Preparation method:
[0197] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.5g potassium nitrate, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0198] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0199] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0200] Add tourmaline powder: Weigh 1.0g of tourmaline powder. Start the high-speed shear disperser (IKA T25, 10000 rpm) and slowly add the tourmaline powder. After all the powder has been added, continue high-speed shearing for 5 minutes to ensure the powder is evenly dispersed.
[0201] Adding liposomes: Stop shearing and resume slow stirring at 300 rpm. Weigh out the equivalent of a liposome suspension containing 0.3 g of propyl gallate and slowly add it dropwise to the system using a dropper. After the addition is complete, continue stirring slowly for 30 minutes.
[0202] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0203] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution, stabilizer solution, and liposome suspension with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0204] Comparative Example 11 (without potassium nitrate)
[0205] Formula (based on 100g):
[0206] Recombinant human ameliorative matrix proteins (rhEMPs): 0.01g, glycerol: 4g, sorbitol: 7g, poloxamer 188: 0.3g, sodium lauroyl sarcosinate: 0.2g, sucralose: 0.005g, xylitol: 0.5g, methylparaben: 0.2g, propylene glycol: 1.0g, PEG-40 hydrogenated castor oil: 0.5g, domiphen: 0.01g, peppermint flavor: 0.05g, purified water: make up to 100g.
[0207] Preparation method:
[0208] Prepare the aqueous phase: Place a clean 250ml beaker on an electronic balance with an accuracy of 0.001g, and weigh in 4g glycerol, 7g sorbitol, 0.5g xylitol, 1.0g propylene glycol, 0.005g sucralose, and 0.01g domiphen. Add approximately 80g of purified water to the beaker. Place the beaker on a magnetic stirrer with a magnetic stir bar, set the speed to 400 rpm, and stir at room temperature until all solids are completely dissolved and the solution becomes clear and transparent.
[0209] Dissolving stabilizers and oily components: Take a 100ml beaker and accurately weigh in 0.3g poloxamer 188, 0.2g sodium lauroyl sarcosinate, 0.5g PEG-40 hydrogenated castor oil, and 0.2g methylparaben. Add 10g purified water to this beaker. Place this small beaker in a 60°C constant temperature water bath and stir continuously with a glass rod until all components are completely dissolved, obtaining a clear solution. Then pour this solution entirely into the large beaker from step 1.
[0210] Adding active protein: Take a 15ml centrifuge tube and accurately weigh 0.01g of rhEMPs dry powder. Add 2ml of purified water to the centrifuge tube, tighten the cap, and vortex for about 1 minute, or until the powder is completely dissolved and the solution is clear. Adjust the stirring speed of the large beaker to 300 rpm, and slowly add the protein solution drop by drop using a dropper or pipette. After all the protein has been added, continue stirring slowly for 15 minutes.
[0211] Adjusting pH: After rinsing the precision pH meter electrode with purified water, immerse it in the solution. Use 0.1M citric acid solution and 0.1M sodium citrate solution to precisely adjust the pH of the system to 7.0.
[0212] Volume adjustment and filling: Add 0.05g of peppermint extract to the solution. Rinse the containers that held the protein solution and stabilizer solution with a small amount of purified water, and add all the rinsing solution to the large beaker. Finally, add purified water to the large beaker until the electronic balance shows a total mass of 100.00g.
[0213] All embodiments and comparative examples were tested according to the following methods, and the results are shown in Table 1.
[0214] Detection method:
[0215] rhEMPs stability (HPLC): Method as before. Calculate protein solubility 24 hours after preparation.
[0216] Inhibition zone test: The paper disc diffusion method was used. Porphyromonas gingivalis (ATCC 33277) was used as the indicator bacterium. Filter paper discs soaked in the sample were placed on agar plates inoculated with bacterial solution and anaerobically incubated at 37°C for 48 hours before measuring the diameter of the inhibition zone.
[0217] Cellular anti-inflammatory assay: An LPS (lipopolysaccharide) inflammation model was constructed using human gingival epithelial cells (HGE cells). After cell treatment with samples, the concentrations of IL-1β and TNF-α in the cell supernatant were detected by ELISA.
[0218] Rat periodontitis model: SD rats were used to establish a periodontitis model using the silk ligation method. Different groups were given appropriate mouthwash treatments. After sacrifice, periodontal tissue pathological scores, alveolar bone resorption (Micro-CT), and IL-1β and TNF-α levels in gingival tissue were measured (ELISA).
[0219] Table 1 Test Results
[0220]
[0221] As can be seen, Comparative Examples 1-4 confirm that the specific poloxamer 188 / sodium lauroyl sarcosinate complex stabilizing system is an absolute prerequisite for the effectiveness of the basic formulation, and its stabilizing and synergistic effects are far superior to those of single components or systems with imbalanced proportions. Secondly, while the effects of Comparative Examples 5-7 and 8-10 were better than Example 3, none reached or even significantly surpassed the level of Example 1, and were far inferior to Example 4. This clearly confirms that there is a strict synergistic dependency between the three advanced components and the formulation; only when all three coexist can an unexpected and superior effect greater than three be produced. No single or dual-component combination can achieve this breakthrough effect. Finally, Example 4 achieved a reduction rate of 55.2% in the key indicator of inhibiting alveolar bone resorption, significantly better than all other groups.
[0222] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0223] The present invention and its embodiments have been described above. This description is not restrictive, and practical applications are not limited thereto. In conclusion, if those skilled in the art are inspired by this description and, without departing from the spirit of the invention, design similar methods and embodiments to this technical solution, all such designs should fall within the protection scope of the present invention.
Claims
1. The application of recombinant human enamel matrix protein in the preparation of anti-periodontal oral care products, characterized by: The anti-periodontalgia oral care product contains the following components: Recombinant human ameliorative matrix protein 0.001-0.1 wt%; glycerol 1-5 wt%; sorbitol 5-10 wt%; poloxamer 188 0.1-0.5 wt%; sodium lauroyl sarcosinate 0.1-0.5 wt%; potassium nitrate 0.1-1 wt%; sucralose 0.001-0.01 wt%; xylitol 0.1-1 wt%; methylparaben 0.1-0.3 wt%; propylene glycol 0.1-1.5 wt%; PEG-40 hydrogenated castor oil 0.1-1 wt%; domiphen 0.005-0.2 wt%; balance: purified water and fragrance.
2. The application according to claim 1, characterized in that: The mass ratio of poloxamer 188 to sodium lauroyl sarcosinate is 1:1 to 1:
2.
3. The application according to claim 1, characterized in that: The pH value of the anti-periodontalgia oral care product is adjusted to 6.5-7.
5.
4. The application according to claim 1, characterized in that: The oral care products for periodontitis are mouthwash, oral spray, or toothpaste.
5. The application according to claim 1, characterized in that: The oral care product for treating periodontitis also contains 0.5-2 wt% sodium hyaluronate.
6. The application according to claim 5, characterized in that: The molecular weight of the sodium hyaluronate is 800,000 to 1,500,000 Daltons.
7. The application according to claim 1, characterized in that: The oral care product for treating periodontitis also contains 0.5-2 wt% of submicron-sized tourmaline powder, the particle size of which is 200-500 nm, and the composition is magnesium iron lithium tourmaline.
8. The application according to claim 1, characterized in that: The oral care product for periodontitis also contains 0.1-0.5 wt% liposome-encapsulated propyl gallate.
9. The application according to claim 8, characterized in that: The method for preparing the liposome-encapsulated propyl gallate includes: dissolving lecithin and cholesterol in anhydrous ethanol at a mass ratio of 3:1, rotary evaporating to form a lipid film, adding a PBS solution containing 0.5% propyl gallate, hydrating at 55°C for 30 min, and extruding through a 0.22 μm filter membrane.