A liquid for repairing oral mucosal lesions and a method for preparing the same

Abstract

The application discloses a liquid for repairing oral mucosa injury and belongs to the technical field of mucosa repair. The liquid comprises seabuckthorn seed oil extract, Periplaneta americana active polypeptide, recombinant human collagen type III, polyoxyethylene 40 hydrogenated castor oil, poloxamer, propylene glycol, glycerol, dipotassium glycyrrhizinate, sucralose, a cooling agent, nipagin methyl ester, nipagin propyl ester, essence, and purified water. The liquid prepared by the application has a smooth and soft taste, a sweet and pleasant flavor, a fresh and clean breath after spraying / flushing, good product stability, no mucosa irritation, and can quickly repair damaged mucosa, and the preparation method is simple, so that the liquid can be used for industrial large-scale production.

Description

Technical Field

[0001] This invention relates to the field of mucosal repair technology, specifically to a liquid for repairing oral mucosal damage and its preparation method. Background Technology

[0002] The oral mucosa is a soft layer of tissue lining the oral cavity. It not only provides protection but also participates in vital physiological functions such as chewing, swallowing, and speech. Oral mucosal injury is a common condition that can be caused by a variety of factors, including trauma, infection, drug side effects, radiation therapy, chemical burns, and autoimmune diseases. Oral mucosal injury can lead to problems such as pain, bleeding, infection, difficulty swallowing, and speech impairment, significantly impacting patients' quality of life. Currently, oral mucosal repair mainly relies on the natural healing process, but this process can be slow and incomplete, especially under certain pathological conditions. Therefore, developing effective compositions that promote oral mucosal repair has significant clinical implications.

[0003] Traditional treatments for oral mucosal repair include medication, physical therapy, and surgery. Medication typically uses anti-inflammatory analgesics, antibiotics, and topical anti-inflammatory drugs, but these medications may have side effects and their effectiveness in promoting mucosal healing is limited. Physical therapy, such as cold light therapy, can relieve symptoms but similarly offers little help in accelerating the healing process. Surgical treatment is usually used when the injury is severe or has not healed for a long time, but it carries certain risks and complications. Furthermore, some existing products that promote healing, such as growth factors and biological dressings, while able to promote healing to some extent, suffer from high costs, strict storage and transportation requirements, and inconvenience in use, limiting their application.

[0004] American cockroach extract has anti-inflammatory, detoxifying, and antibacterial properties. It is typically obtained as a brown powder by crushing the cockroach body, adding ethanol, heating under normal pressure, and then drying. Technically, this is only a crude extract with many impurities, low purity, and low stability, making content testing and quality control impossible. Currently, it is not used for wound healing, and even if it were, product quality would be difficult to guarantee. Therefore, designing a product based on American cockroaches that promotes mucosal injury healing would be beneficial to the development of mucosal repair technology. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention presents a liquid for repairing oral mucosal damage and its preparation method.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0007] On one hand, the present invention provides a liquid for repairing oral mucosal damage. By weight, the liquid comprises 0.05-0.6 parts of sea buckthorn seed oil extract, 0.005-0.06 parts of American cockroach active polypeptide, 0.005-0.06 parts of recombinant type III human collagen, 9-15 parts of polyoxyethylene 40 hydrogenated castor oil, 0.005-0.06 parts of poloxamer, 20-40 parts of propylene glycol, 80-100 parts of glycerol, 0.02-0.2 parts of dipotassium glycyrrhizate, 0.1-0.3 parts of sucralose, 0.1-0.5 parts of cooling agent, 0.02-0.2 parts of methylparaben, 0.1-0.8 parts of propylparaben, 0.5-5 parts of fragrance, and 800-900 parts of water.

[0008] Preferably, by weight, the liquid comprises 0.1-0.5 parts of sea buckthorn seed oil extract, 0.01-0.05 parts of American cockroach active polypeptide, 0.01-0.05 parts of recombinant type III human collagen, 11-13 parts of polyoxyethylene 40 hydrogenated castor oil, 0.01-0.05 parts of poloxamer, 25-35 parts of propylene glycol, 85-95 parts of glycerol, 0.08-0.12 parts of dipotassium glycyrrhizate, 0.15-0.2 parts of sucralose, 0.25-0.3 parts of cooling agent, 0.08-0.12 parts of methylparaben, 0.4-0.6 parts of propylparaben, 1-3 parts of fragrance, and 876.19-852.01 parts of water.

[0009] More preferably, by weight, the liquid comprises 0.3 parts sea buckthorn seed oil extract, 0.03 parts American cockroach active polypeptide, 0.03 parts recombinant type III human collagen, 12 parts polyoxyethylene 40 hydrogenated castor oil, 0.03 parts poloxamer, 30 parts propylene glycol, 90 parts glycerol, 0.1 parts dipotassium glycyrrhizate, 0.18 parts sucralose, 0.27 parts cooling agent, 0.1 parts methylparaben, 0.5 parts propylparaben, 2 parts fragrance, and 865 parts water.

[0010] Preferably, the liquid is a spray or a gargle solution.

[0011] Preferably, the preparation of the sea buckthorn seed oil extract includes: first, making sea buckthorn seeds into powder of 20-40 mesh, and then extracting them in a supercritical carbon dioxide extraction vessel for 1-3 hours to obtain the sea buckthorn seed oil extract.

[0012] More preferably, the supercritical carbon dioxide extraction conditions are: carbon dioxide flow rate of 20~35 L / h, extraction vessel temperature of 45~55℃, extraction pressure of 25~35 MPa, and separation vessel temperature of 20~30℃.

[0013] Preferably, the active peptide from the American cockroach is prepared using a bioaffinity purification method, specifically including the following steps:

[0014] S1. The dried American cockroach protozoa were crushed, and then 0.01 M PBS was added at a ratio of 1:(10~20). The mixture was placed at room temperature for 8~12 h, and then subjected to three freeze-thaw cycles. After centrifugation at 10000×g for 15 min, the supernatant was collected and precipitated with 50% sodium sulfate. After standing at 4℃ for 20 h, the mixture was centrifuged for 20 min. The precipitate was dissolved in purified water and dialyzed for 24 h to remove salt, and then the American cockroach polypeptide extract was obtained.

[0015] S2. Active peptides from American cockroaches were separated using reversed-phase high-performance liquid chromatography.

[0016] More preferably, in step S2, mobile phase A is water containing 0.1% trifluoroacetic acid; and mobile phase B is 2-propanol containing 0.1% TFA.

[0017] Furthermore, the present invention also provides a method for preparing the aforementioned liquid for repairing oral mucosal damage, comprising the following steps:

[0018] Step 1: Add purified water to the preparation vessel, then add glycerin and stir well. Then, disperse and dissolve the active peptides of American cockroaches in purified water and add them to the preparation vessel, stirring until well mixed.

[0019] Step 2: Dissolve dipotassium glycyrrhizate and sucralose in purified water to obtain a sweet solution, pour it into a mixing tank, and continue stirring;

[0020] Step 3: Dissolve recombinant type III human collagen in purified water and pour it into the preparation container, then continue stirring;

[0021] Step 4: Mix propylene glycol with RH-40 and poloxamer until well combined. Then add methylparaben, propylparaben, and cooling agent and dissolve completely. Add fragrance and mix well. Pour into a preparation container and continue stirring. Then slowly add sea buckthorn seed oil extract to the preparation container and stir until well combined. This gives you a semi-finished liquid for repairing oral mucosal damage. Bottle the product to obtain the liquid for repairing oral mucosal damage.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention provides a liquid for repairing oral mucosal damage. The liquid comprises sea buckthorn seed oil extract, American cockroach active peptides, recombinant type III human collagen, polyoxyethylene 40 hydrogenated castor oil, poloxamer, propylene glycol, glycerol, dipotassium glycyrrhizate, sucralose, a cooling agent, methylparaben, propylparaben, flavoring, and purified water. The liquid prepared by this invention has a mild and smooth taste, a sweet and pleasant flavor, and leaves breath feeling fresh and clean after spraying / rinsing. It exhibits good product stability and does not irritate the mucosa.

[0024] The bioactive peptides extracted from the American cockroach in this invention are of high purity, low impurities, and high stability. They can promote the repair of damaged oral mucosa, soothe gingival swelling and pain, improve the oral environment, reduce plaque buildup, and reduce the occurrence of oral problems such as tooth decay, periodontitis, and gingivitis. Sea buckthorn seed oil is rich in unsaturated fatty acids, carotenoids, natural tocopherols (VE), flavonoids, and ergosterol, which have significant antioxidant activity. It is also very helpful in relieving pain caused by damage and has a definite effect on repairing skin and mucosal damage and controlling and improving inflammation in damaged areas. This invention, through the synergistic effect of its components, can effectively promote mucosal healing. The preparation method is simple, highly reproducible, and suitable for large-scale industrial production. Attached Figure Description

[0025] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a graph showing the statistical values ​​of the number of test colonies after using phosphate buffer in the non-metabolic activation assay for the toxicity detection of active peptides in American cockroaches.

[0027] Figure 2 This is a graph showing the statistical values ​​of the number of test colonies after using the S9 mixture in the non-metabolic activation test for the toxicity detection of active peptides in American cockroaches.

[0028] Figure 3 This is a statistical chart of the number of individual colonies in the toxicity test of active polypeptides in American cockroaches.

[0029] Figure 4 This is a statistical chart of the number of colonies in the first test of the toxicity detection of active peptides in American cockroaches.

[0030] Figure 5 This is a diagram showing the results of an acute eye irritation / corrosion test on active peptides from the American cockroach.

[0031] Figure 6 This is a diagram showing the results of the micronucleus assay of mouse bone marrow polychromatic erythrocytes with active peptides from American cockroaches.

[0032] Figure 7 This is a graph showing the cell activity test results of active peptides from the American cockroach.

[0033] Figure 8 This is a bar chart showing the relative activity of active polypeptides from American cockroaches in different cells.

[0034] Figure 9 This is a graph showing the cell scratch test results of active peptides from the American cockroach. The numerical values ​​in the graph are in units of .

[0035] Figure 10 This is a comparison chart of the relative areas of scratches on active polypeptides from the American cockroach.

[0036] Figure 11 This is a microscope image of the control group in the cell scratch experiment.

[0037] Figure 12 These are microscope images of different concentrations of active peptides from American cockroaches in a cell scratch assay. Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0040] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0041] The cooling agent used in the following examples is a menthol derivative, WS-3; all other test methods or materials used are conventional methods or reagents and materials that are commercially available.

[0042] Example 1

[0043] I. A liquid for repairing oral mucosal damage, comprising, by weight parts: 0.3 parts sea buckthorn seed oil extract, 0.03 parts American cockroach active polypeptide, 0.03 parts recombinant type III human collagen, 12 parts polyoxyethylene 40 hydrogenated castor oil (RH-40), 0.03 parts poloxamer, 30 parts propylene glycol, 90 parts glycerol, 0.1 parts dipotassium glycyrrhizate, 0.18 parts sucralose, 0.27 parts cooling agent, 0.1 parts methylparaben, 0.5 parts propylparaben, 2 parts fragrance, and 865 parts purified water.

[0044] The preparation of the sea buckthorn seed oil extract is as follows: sea buckthorn seeds are dried at 65°C and then pulverized into 30-mesh powder. Then, 500 g of the powder is placed in a supercritical carbon dioxide extraction vessel (carbon dioxide flow rate of 25 L / h, extraction vessel temperature of 50°C, extraction pressure of 30 MPa, and separation vessel temperature of 30°C) for 2 h to obtain the sea buckthorn seed oil extract.

[0045] The active peptide from the American cockroach was prepared using a bioaffinity purification method, specifically including the following steps:

[0046] (1) Weigh 500 g of dried American cockroach protozoa and crush them. Then add 0.01 M PBS (pH 7.0) at a ratio of 1:15 and place at room temperature for 10 h. After repeated freeze-thaw cycles three times, centrifuge at 10000×g for 15 min. Take the supernatant and add 50% sodium sulfate to precipitate. After standing at 4℃ for 20 h, centrifuge for 20 min (4℃, 12000×g). Dissolve the precipitate in purified water and dialyze for 24 h to remove salt to obtain American cockroach polypeptide extract.

[0047] (2) Separation using reversed-phase high-performance liquid chromatography

[0048] Column: Zorbax SB-C18 (4.6 mm × 250 mm, 5 μm);

[0049] Mobile phase: Phase A: water containing 0.1% trifluoroacetic acid (TFA); Phase B: 2-propanol containing 0.1% TFA; Detector: DAD; Detection wavelength: 230 nm. The high-activity peak obtained by gel column chromatography elution was separated by RP-HPLC using stepwise elution. Elution was performed first with eluent A for 6 min, followed by eluent B for 10 min. The flow rate was 2 mL / min. The corresponding fractions were collected and freeze-dried to obtain the active peptides of American cockroach.

[0050] II. According to the above formula, the specific method for preparing the liquid is as follows:

[0051] 1. Add purified water to the preparation vessel, then add glycerin and stir well. Then, disperse and dissolve the active peptides of American cockroaches in purified water and add them to the preparation vessel, stirring until well mixed.

[0052] 2. Dissolve dipotassium glycyrrhizate and sucralose in purified water to obtain a sweet solution, pour it into a mixing tank, and continue stirring.

[0053] 3. Dissolve the recombinant type III human collagen in purified water and pour it into the preparation container, then continue stirring.

[0054] 4. Mix propylene glycol with RH-40 and poloxamer until well combined. Then add methylparaben, propylparaben, and cooling agent until completely dissolved. Add flavor (menthol), mix well, and pour into a preparation container. Continue stirring. Then slowly add sea buckthorn seed oil extract to the preparation container and stir until well combined. This gives you a semi-finished liquid product for repairing oral mucosal damage.

[0055] 5. Dispense the semi-finished liquid into suitable containers, seal them, and then transfer them to the outer packaging process for bottling.

[0056] Example 2

[0057] The difference between Example 2 and Example 1 is that the formulation of the liquid for repairing oral mucosal damage is different, but everything else is the same.

[0058] A liquid for repairing oral mucosal damage, comprising, by weight parts: 0.1 parts sea buckthorn seed oil extract, 0.03 parts American cockroach active polypeptide, 0.05 parts recombinant type III human collagen, 11 parts polyoxyethylene 40 hydrogenated castor oil, 0.03 parts poloxamer, 35 parts propylene glycol, 90 parts glycerol, 0.08 parts dipotassium glycyrrhizate, 0.18 parts sucralose, 0.25 parts cooling agent, 0.1 parts methylparaben, 0.5 parts propylparaben, 1 part fragrance, and 876.19 parts purified water.

[0059] Example 3

[0060] The difference between Example 3 and Example 1 is that the formulation of the liquid for repairing oral mucosal damage is different, but everything else is the same.

[0061] A liquid for repairing oral mucosal damage, comprising, by weight parts: 0.3 parts sea buckthorn seed oil extract, 0.01 parts American cockroach active polypeptide, 0.03 parts recombinant type III human collagen, 12 parts polyoxyethylene 40 hydrogenated castor oil, 0.01 parts poloxamer, 30 parts propylene glycol, 85 parts glycerol, 0.12 parts dipotassium glycyrrhizate, 0.18 parts sucralose, 0.27 parts cooling agent, 0.12 parts methylparaben, 0.5 parts propylparaben, 3 parts fragrance, and 865 parts purified water.

[0062] Example 4

[0063] Example 4 differs from Example 1 in that the formulation of the liquid for repairing oral mucosal damage is different, but everything else is the same.

[0064] A liquid for repairing oral mucosal damage, comprising, by weight parts: 0.5 parts sea buckthorn seed oil extract, 0.03 parts American cockroach active polypeptide, 0.03 parts recombinant type III human collagen, 13 parts polyoxyethylene 40 hydrogenated castor oil, 0.03 parts poloxamer, 25 parts propylene glycol, 90 parts glycerol, 0.1 parts dipotassium glycyrrhizate, 0.2 parts sucralose, 0.27 parts cooling agent, 0.08 parts methylparaben, 0.6 parts propylparaben, 2 parts fragrance, and 865 parts purified water.

[0065] Example 5

[0066] Example 5 differs from Example 1 in that the formulation of the liquid for repairing oral mucosal damage is different, but everything else is the same.

[0067] A liquid for repairing oral mucosal damage, comprising, by weight parts: 0.3 parts sea buckthorn seed oil extract, 0.05 parts American cockroach active polypeptide, 0.01 parts recombinant type III human collagen, 12 parts polyoxyethylene 40 hydrogenated castor oil, 0.05 parts poloxamer, 30 parts propylene glycol, 95 parts glycerol, 0.1 parts dipotassium glycyrrhizate, 0.15 parts sucralose, 0.3 parts cooling agent, 0.1 parts methylparaben, 0.4 parts propylparaben, 2 parts fragrance, and 852.01 parts purified water.

[0068] Example 6

[0069] The difference between Example 6 and Example 1 is that the preparation of the sea buckthorn seed oil extract is different, but everything else is the same.

[0070] Preparation of the sea buckthorn seed oil extract: First, the sea buckthorn seeds are dried at 65℃ and then pulverized into 20-mesh powder. Then, 500 g of the powder is placed in a supercritical carbon dioxide extraction vessel (carbon dioxide flow rate of 30 L / h, extraction vessel temperature of 55℃, extraction pressure of 25 MPa, and separation vessel temperature of 30℃) for 1 h to obtain the sea buckthorn seed oil extract.

[0071] Example 7

[0072] The difference between Example 7 and Example 1 is that the preparation of the sea buckthorn seed oil extract is different, but everything else is the same.

[0073] Preparation of the sea buckthorn seed oil extract: First, the sea buckthorn seeds are dried at 65℃ and then pulverized into 40-mesh powder. Then, 500 g of the powder is placed in a supercritical carbon dioxide extraction vessel (carbon dioxide flow rate of 20 L / h, extraction vessel temperature of 45℃, extraction pressure of 35 MPa, and separation vessel temperature of 20℃) for 3 h to obtain the sea buckthorn seed oil extract.

[0074] Comparative Example 1

[0075] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain sea buckthorn seed oil extract, but everything else is the same.

[0076] Comparative Example 2

[0077] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain the American cockroach bioactive peptide, but all other aspects are the same.

[0078] Comparative Example 3

[0079] The difference between Comparative Example 3 and Example 1 is that the bioactive peptides of American cockroaches were replaced with ethanol extracts of dried American cockroach bodies in Comparative Example 3, while all other aspects were the same.

[0080] Comparative Example 4

[0081] The difference between Comparative Example 4 and Example 1 is that Comparative Example 4 does not contain recombinant type III human collagen, but all other aspects are the same.

[0082] Comparative Example 5

[0083] The difference between Comparative Example 5 and Example 1 is that the preparation of the sea buckthorn seed oil extract is different, but everything else is the same.

[0084] Preparation of the sea buckthorn seed oil extract: First, the sea buckthorn seeds are dried at 65℃ and then pulverized into 20-mesh powder. Then, 500 g of the powder is placed in a supercritical carbon dioxide extraction vessel (carbon dioxide flow rate of 40 L / h, extraction vessel temperature of 35℃, extraction pressure of 15 MPa, and separation vessel temperature of 35℃) for 0.5 h to obtain the sea buckthorn seed oil extract.

[0085] Comparative Example 6

[0086] The difference between Comparative Example 6 and Example 1 is that the preparation of the sea buckthorn seed oil extract is different, but everything else is the same.

[0087] Preparation of the sea buckthorn seed oil extract: First, the sea buckthorn seeds are dried at 65℃ and then pulverized into 20-mesh powder. Then, 500 g of the powder is placed in a supercritical carbon dioxide extraction vessel (carbon dioxide flow rate of 10 L / h, extraction vessel temperature of 65℃, extraction pressure of 45 MPa, and separation vessel temperature of 15℃) for 3 h to obtain the sea buckthorn seed oil extract.

[0088] Test Example 1

[0089] I. The toxicity of the American cockroach active polypeptide prepared in Example 1 was tested in accordance with the "Cosmetic Safety Technical Specifications" (2015).

[0090] 1.1 Test strains: Histidine-deficient strains of Salmonella Typhimurium TA97, TA98, TA100 and TA102 were purchased from KRISHGEN BioSystems and CHI SCIENTIFIC.

[0091] 1.2 Metabolite Activation System: S9

[0092] 1.3 Positive reagents: Sodium azide (NaN3) was purchased from Sinopharm Chemical Reagent Co., Ltd. (batch number: 20180321), Dexon was purchased from AccuStandard (batch number: 4030165-01), 2-aminofluorene (2-AF) was purchased from aladdin (batch number: H1511011), 1,8-dihydroxyanthraquinone (Dorbane) was purchased from Sigma-Aldrich (batch number: WXBB0392), and 2-aminoanthracene (2-AA) was purchased from Sigma-Aldrich (batch number: STBD3302V).

[0093] 1.4 Culture conditions: 37℃ incubator

[0094] 2. Experimental Procedure:

[0095] 2.1 Bacterial Preparation: Before the experiment, the bacterial strain was inoculated into nutrient broth and incubated statically at approximately 37°C in a constant temperature incubator for nearly 16 hours. This was to allow the bacterial count to reach approximately 1 × 10⁻⁶ per milliliter. 9 .

[0096] 2.2 Preparation of test substances: Using sterile ultrapure water as solvent, different concentrations of exposure solutions were prepared, namely 50 mg / mL, 15.81 mg / mL, 5 mg / mL and 1.58 mg / mL.

[0097] 2.3 Grouping: Four different dose groups were set up for metabolic activation and non-metabolic activation treatment, and solvent control group, positive control group, sterile control group and blank control group were set up respectively.

[0098] 2.4 Exposure time: 48 hours

[0099] 2.5 Plate doping method:

[0100] Non-metabolic activation: Dispense 2.0 mL of the top agar medium into test tubes and incubate at 45°C. Then, add 0.1 mL of the test strain enrichment broth, 0.1 mL of the test substance solution, and 0.5 mL of phosphate buffer to each tube sequentially, mix thoroughly, and quickly pour onto the bottom medium plate. Rotate the plate to ensure even distribution. Three replicates are set up for all dosage groups and control groups. After the plates are placed horizontally to solidify, invert them and incubate at 37°C for 48 hours. After incubation, observe the background colony growth and record the number of revertant colonies per plate. Metabolic activation system: Replace the 0.5 mL phosphate buffer in the non-metabolic activation test with S9 mixture; other procedures are the same as in the non-metabolic activation test.

[0101] Depend on Figures 1-4 It can be seen that, regardless of whether the metabolism is activated or not, there is no obvious bacterial toxicity in each dose group compared with the solvent control group. The number of revertant mutant colonies of all test strains at each dose is less than twice that of the solvent control group. According to the judgment criteria of the "Cosmetic Safety Technical Specifications" (2015), the test samples are not mutagenic to the test strains.

[0102] II. Acute eye irritation / corrosion test

[0103] 1. Sample: Dilute the sample to a 5% aqueous solution.

[0104] 2. Laboratory animals and environment:

[0105] Common grade New Zealand rabbits, sourced from Guangzhou Huadu District Huadong Xinhua Experimental Animal Farm SCXK (Guangdong) 2019-0023. Environment: Temperature (T): 18~25℃, Relative Humidity (%RH): 40~70%.

[0106] 3. Experimental Procedure:

[0107] Twenty-four hours prior to the experiment, both eyes of the experimental animals underwent routine examination. During the experiment, the lower eyelid of one eye was gently pulled back, and 0.1 mL of the test substance was instilled into the conjunctival sac. The eye was passively closed for 1 second, and the eye was not rinsed within 24 hours after instillation. The other eye was left untreated as a self-control. Eyes were examined and scored at 1 hour, 24 hours, 48 ​​hours, 72 hours, 4 days, 7 days, 14 days, and 21 days after the experiment. If no irritation response was observed within 72 hours, or if the eye had fully recovered by day 7 or 14, the experiment could be terminated early. If necessary, the cornea was examined with 2% sodium fluorescein.

[0108] 4. Results evaluation: Stimulus response scores were calculated based on the cornea, iris, or conjunctiva scores of the test animals at 24 h, 48 h, and 72 h observation points. The ocular irritation response was graded according to Table 1 based on the stimulus response scores and recovery time.

[0109] Table 1. Product Eye Irritation Rating Table

[0110]

[0111] Note: When the corneal, iris, and conjunctival integrals are 0, it can be judged as non-irritating.

[0112] Depend on Figure 5 It can be seen that the active peptides of American cockroaches showed no irritation in the acute eye irritation test on New Zealand rabbits.

[0113] III. The active peptides of American cockroach were subjected to mouse bone marrow polychromatic erythrocyte micronucleus test in accordance with the "Cosmetic Safety Technical Specifications" (2015).

[0114] Animal grouping: After the KM mice had completed their adaptation period, healthy mice were selected and randomly divided into 5 groups according to their weight and sex using a stratified randomization method, with 10 mice in each group and half male and half female.

[0115] Depend on Figure 6 According to the criteria of the "Cosmetic Safety Technical Specifications" (2015), the active peptide of American cockroach did not induce an increase in the incidence of micronuclei in bone marrow PCE, so the result of this test was negative.

[0116] IV. Human gingival fibroblast (HGF) scratch test

[0117] 1. Experimental materials:

[0118] 1.1 Cell line: Human gingival fibroblasts (HGF), purchased from BeiNa ChuangLian, passage number: 6

[0119] 1.2 Complete culture medium: DMEM medium containing 10% fetal bovine serum (FBS) (FBS purchased from Gibco, Lot: 42Q1095K)

[0120] 1.3 Maintenance medium: DMEM medium containing 1% FBS (FBS purchased from Gibco, Lot: 42Q1095K)

[0121] 1.4 Test conditions: Temperature 37±0.5℃, humidity >90%, CO2 concentration 5%.

[0122] 1.5 Solutions and Controls:

[0123] Negative control group (NC): Maintenance culture medium

[0124] Positive control group (PC): maintenance medium containing 1 ng / mL EGF

[0125] Analyte (TA): Weigh 0.2153 g of sample, add 2.15 mL of maintenance medium to prepare a stock solution of 100 mg / mL, and then dilute with maintenance medium to the target concentration.

[0126] 2. Test steps:

[0127] 2.1 Cell viability test

[0128] 2.1.1 Routine cell culture. The cell density was 5–7 × 10⁻⁶. 4 A cell suspension of 100 µL per well was seeded into a 96-well cell culture plate and cultured for 18–24 h.

[0129] 2.1.2 Discard the original culture medium in the wells, add 100 µL of test samples of different concentrations to each well, and return to the incubator for incubation for 48±1 h.

[0130] 2.1.3 Remove the culture plate, add 20 µL of MTT solution to each well, and incubate in an incubator for 3–4 h. Remove the liquid from the wells, add 100 µL of DMSO to each well, and shake on a shaker for 10–15 min. Measure the absorbance at 570 nm using a microplate reader.

[0131] 2.1.4 Data Analysis: Cell viability was calculated with the cell viability of the negative control group as 100%. The relative cell viability of each group was calculated.

[0132] 2.2 Cell Scratch Test

[0133] 2.2.1 Culture cells using standard methods, adjust the cell density to 3.0–5.0 × 10⁵ cells / mL, use a 24-well cell culture plate with a 2-well insert, seed 70 µL in each well, and return to the incubator to culture for 18–24 h until cell confluence.

[0134] 2.2.2 Carefully remove the culture insert from the culture plate using tweezers, ensuring a smooth, cell-free area is formed between the two wells of the culture insert. Wash the cell layer three times with PBS to remove any detached cells.

[0135] 2.2.3 Add 1 mL of culture medium containing different concentrations of test samples to each well, and simultaneously acquire images under a microscope, record them as 0 h, and mark the acquisition location.

[0136] 2.2.4 Every 24 hours, images are collected at the same marked location, recorded as 24 h and 48 h.

[0137] 2.2.5 Data Analysis: IPP image analysis software was used to analyze the area of ​​the scratch region in each image, with 0 h as 100%, and the relative area was calculated.

[0138] For the cell scratch assay, 4.0 mg / mL, 1.0 mg / mL, and 0.25 mg / mL were selected as the concentrations for subsequent efficacy experiments.

[0139] Depend on Figures 7-12 It was found that at 24 h, compared with the negative control group, the relative area of ​​scratches in the positive control group was significantly reduced (P<0.05), and the relative area of ​​scratches in the samples at concentrations of 4.0 mg / mL, 1.0 mg / mL, and 0.25 mg / mL was significantly reduced (P<0.05). At 48 h, compared with the negative control group, the relative area of ​​scratches in the positive control group was significantly reduced (P<0.05), and the relative area of ​​scratches in the samples at concentrations of 4.0 mg / mL, 1.0 mg / mL, and 0.25 mg / mL was significantly reduced (P<0.05). This suggests that the samples have a certain promoting effect on the repair of human gingival fibroblasts.

[0140] Test Example 2

[0141] Subjects were selected to rinse their mouths twice daily, morning and evening, using 25 mL of the liquids prepared in Examples 1-7 and Comparative Examples 1-6, respectively, to test the effectiveness in eliminating bad breath, preventing tooth decay, and promoting oral mucosal healing for 30 days. Subjects participating in the tests for eliminating bad breath, preventing tooth decay, and promoting oral mucosal healing also provided sensory evaluations, such as flavor, sweetness, appearance, and packaging quality. The evaluation criteria for the test results are shown in Table 2 below. The effectiveness of the test results is expressed as a percentage.

[0142] Table 2 Test Evaluation Criteria

[0143]

[0144] Table 3 Test Results

[0145]

[0146] As shown in Tables 2-3, compared with Comparative Examples 1-6, the liquids prepared in Examples 1-7 effectively promoted oral mucosal healing, and their taste, flavor, aroma, sweetness, and appearance were all satisfactory. Only liquids prepared by combining and complementing each other within appropriate ranges and preparation conditions, containing sea buckthorn seed oil extract, American cockroach active peptides, recombinant type III human collagen, polyoxyethylene 40 hydrogenated castor oil, poloxamer, propylene glycol, glycerol, dipotassium glycyrrhizate, sucralose, cooling agent, methylparaben, propylparaben (0.1-0.8 parts), flavoring, and purified water, possess good taste, sweetness, and effectively promote the repair of oral mucosal damage, reducing the pain and recovery time for patients with oral ulcers and other mucosal injuries, and helping to combat mucosal damage problems.

[0147] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A liquid for repairing oral mucosa damage, characterized by comprising, The liquid comprises, by mass fraction, 0.05-0.6 parts of seabuckthorn seed oil extract, 0.005-0.06 parts of active polypeptide of Periplaneta americana, 0.005-0.06 parts of recombinant human collagen type III, 9-15 parts of polyoxyethylene 40 hydrogenated castor oil, 0.005-0.06 parts of poloxamer, 20-40 parts of propylene glycol, 80-100 parts of glycerol, 0.02-0.2 parts of dipotassium glycyrrhizinate, 0.1-0.3 parts of sucralose, 0.1-0.5 parts of cooling agent, 0.02-0.2 parts of methyl nipagin, 0.1-0.8 parts of propyl nipagin, 0.5-5 parts of essence, and 800-900 parts of water; The preparation of the seabuckthorn seed oil extract comprises: first, preparing seabuckthorn seed powder with a particle size of 20-40 microns, and then placing the powder in a supercritical carbon dioxide extraction kettle for extraction for 1-3 hours to obtain the seabuckthorn seed oil extract; The supercritical carbon dioxide extraction conditions are as follows: the carbon dioxide flow rate is 20-35 L / h, the extraction kettle temperature is 45-55℃, the extraction pressure is 25-35 MPa, and the separation kettle temperature is 20-30℃; The active polypeptide of Periplaneta americana is prepared by bioaffinity purification, and the preparation process comprises the following steps: S1. The dried Periplaneta americana powder is crushed, then 1: (10-20) of 0.01M PBS is added and placed at room temperature for 8-12 hours, then repeatedly frozen and thawed three times, centrifuged at 10,000xg for 15 minutes, and the supernatant is added with 50% sodium sulfate for salting out, and then centrifuged at 4℃ for 20 hours, and the precipitate is dissolved in purified water and dialyzed for 24 hours to remove salt to obtain the Periplaneta americana polypeptide extract; S2. The active polypeptide of Periplaneta americana is separated by reverse-phase high-performance liquid chromatography.

2. The liquid for repairing oral mucosa damage according to claim 1, wherein The liquid comprises, by mass fraction, 0.1-0.5 parts of seabuckthorn seed oil extract, 0.01-0.05 parts of active polypeptide of Periplaneta americana, 0.01-0.05 parts of recombinant human collagen type III, 11-13 parts of polyoxyethylene 40 hydrogenated castor oil, 0.01-0.05 parts of poloxamer, 25-35 parts of propylene glycol, 85-95 parts of glycerol, 0.08-0.12 parts of dipotassium glycyrrhizinate, 0.15-0.2 parts of sucralose, 0.25-0.3 parts of cooling agent, 0.08-0.12 parts of methyl nipagin, 0.4-0.6 parts of propyl nipagin, 1-3 parts of essence, and 852.01-876.19 parts of water.

3. The liquid for repairing oral mucosa damage according to claim 1, wherein The liquid comprises, by mass fraction, 0.3 parts of seabuckthorn seed oil extract, 0.03 parts of active polypeptide of Periplaneta americana, 0.03 parts of recombinant human collagen type III, 12 parts of polyoxyethylene 40 hydrogenated castor oil, 0.03 parts of poloxamer, 30 parts of propylene glycol, 90 parts of glycerol, 0.1 parts of dipotassium glycyrrhizinate, 0.18 parts of sucralose, 0.27 parts of cooling agent, 0.1 parts of methyl nipagin, 0.5 parts of propyl nipagin, 2 parts of essence, and 865 parts of water.

4. The liquid for repairing oral mucosa damage according to claim 1, wherein The liquid is a spray or a gargle.

5. The liquid for repairing oral mucosa damage according to claim 1, wherein In step S2, the mobile phase A is water containing 0.1% trifluoroacetic acid, and the mobile phase B is 2-propanol containing 0.1% TFA.

6. A method for preparing a liquid for repairing lesions of the oral mucosa according to any one of claims 1 to 5, characterized in that The method comprises the following steps: Step 1: Add purified water to the preparation vessel, then add glycerol and stir well. Then disperse and dissolve the active peptides of American cockroaches in purified water and add them to the preparation vessel, stirring until well mixed. Step 2: Dissolve dipotassium glycyrrhizate and sucralose in purified water to obtain a sweet solution, pour it into a mixing tank, and continue stirring; Step 3: Dissolve recombinant type III human collagen in purified water and pour it into the preparation container, then continue stirring; Step 4: Mix propylene glycol with polyoxyethylene 40 hydrogenated castor oil and poloxamer until well combined. Then add methylparaben, propylparaben, and cooling agent and dissolve completely. Add fragrance and mix well. Pour into a preparation container and continue stirring. Then slowly add sea buckthorn seed oil extract to the preparation container and stir until well combined. This gives you a semi-finished liquid for repairing oral mucosal damage. Bottle the product to obtain the liquid for repairing oral mucosal damage.

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