Preparation method of oral collagen base toothpaste raw material and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method of a collagen dental base paste raw material for oral cavity and application thereof. The steps are as follows: firstly, dissolving recombinant collagen in water for injection to form a collagen solution; then adding hydroxyapatite and an aqueous solvent into an oil phase solvent, stirring uniformly to obtain a hydroxyapatite suspension; then preparing an EDC solution and an NHS solution, mixing the two, and adding the mixture into the collagen solution to obtain a collagen mixture; then adding the hydroxyapatite suspension, and after ultrasonic treatment, centrifugation and natural air drying, the collagen dental base paste raw material for oral cavity is obtained. The application uses the combination of the aqueous solvent and the oil phase solvent, so that the Hap and the collagen can better form a polymer; the use of the collagen, the NHS and the EDC can improve the antibacterial performance; and the final product is in the form of powder (nanoscale), has delicate texture, can strengthen the effects of hydroxyapatite remineralization and desensitization, and realizes the combination of gum protection, caries prevention and anti-inflammatory function.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing collagen-based toothpaste raw materials for oral use and its application. Background Technology

[0002] Toothpaste is one of the most commonly used daily chemical products. Related studies show that each person needs to use 1.7 to 2.2 grams of toothpaste daily, which translates to 620 to 800 tons of toothpaste consumed annually per million people. In 2020, my country's toothpaste production was approximately 7 billion tubes (based on 100 grams per tube), making it the world's largest producer, consumer, and exporter of toothpaste. With the development of the times and the improvement of people's living standards, the purpose of using toothpaste is no longer just for cleaning the mouth; people are increasingly pursuing oral health while maintaining oral hygiene.

[0003] Unhealthy eating habits and lifestyle changes in daily life, such as staying up late, eating spicy food, and a fast-paced lifestyle, can lead to varying degrees of oral health problems. Tooth decay and dentin hypersensitivity are common oral health issues that have received increasing attention in recent years. Since teeth are located on the gums, which contain many dental nerves and blood vessels, and are constantly in contact with external stimuli, bacteria can easily invade and cause disease. Tooth decay is a common dental disease, also known as cavities or tooth decay. This is caused by bacteria in the mouth invading the teeth, damaging the tooth tissue, causing nerve pain, and creating cavities. After developing tooth decay, the nerve pain in the teeth can irritate the patient, making it difficult to maintain normal movement. When eating, food touching the damaged tissue or cavities can also irritate the dental nerves, causing pain.

[0004] Dentin hypersensitivity, also known as sensitive dentin, or commonly called "teeth sensitivity," refers to toothache triggered by contact with cold, hot, sour, or sweet foods or external stimuli such as temperature changes. Many factors can cause dentin hypersensitivity, including damage to the enamel and exposure of the dentin, such as mechanical wear, acid erosion, wedge-shaped defects, cracks, tooth fractures, dental caries, and periodontal recession leading to exposure of the tooth neck. Epidemiological surveys show that approximately 29.7% of adults in my country experience varying degrees of dentin hypersensitivity.

[0005] To prevent these oral health problems, maintaining good oral hygiene is paramount. Daily oral hygiene includes brushing your teeth regularly and rinsing your mouth appropriately to ensure no food residue remains and prevent bacterial growth. While there are many treatments for cavities and dentin hypersensitivity, only about 5% of patients seek treatment at a dentist. Therefore, functional toothpastes that can be used at home have become increasingly popular.

[0006] Toothpastes on the market are becoming increasingly specialized, and single-function toothpastes can no longer meet people's growing needs. Therefore, it is urgent to add effective ingredients to toothpastes that provide whitening and cleaning benefits, giving them additional advantages such as cavity prevention, dentin hypersensitivity relief, gum protection, and heat relief.

[0007] Synthetic hydroxyapatite is a good toothpaste additive with excellent biocompatibility. It can be applied directly to the tooth surface, blending with the hydroxyapatite naturally present in teeth and even repairing minor cracks on the tooth surface. Adding hydroxyapatite to toothpaste and other dental cleaning products can stimulate and activate periodontal tissues, offering unique therapeutic effects in tooth remineralization, treatment of tooth sensitivity, and antibacterial activity, thus preventing gingivitis and periodontal disease. However, the effects of hydroxyapatite are relatively limited and its effects are slow to appear.

[0008] Remineralization is primarily influenced by the excellent biocompatibility and bioactivity of hydroxyapatite. The tooth surface is covered by a 1-2 nm thick layer of enamel, whose main component is also hydroxyapatite. Food debris in the mouth decomposes into acidic substances under the action of bacteria, which can corrode the hydroxyapatite on the tooth surface, leading to demineralization and creating pores of varying sizes on the tooth surface. These damaged areas are prone to plaque buildup and bacterial invasion, eventually causing tooth decay and other oral diseases. Hydroxyapatite remineralization involves the reformation of hydroxyapatite crystals at the site of tooth decay, maintaining the balance of calcium and phosphorus ions in saliva and thus preventing tooth demineralization. However, hydroxyapatite itself does not possess antibacterial properties, and its effectiveness when used alone is limited. Currently, functional toothpastes on the market are generally used in combination with medications to form medicated toothpastes. During brushing, the medication reduces plaque, thus preventing tooth decay and periodontal disease. However, medicated toothpaste is easily rinsed out of the mouth in a short time, making it difficult to achieve its intended effect. Furthermore, the use of medication can irritate the soft tissues in the mouth, leading to some side effects. Summary of the Invention

[0009] To address the shortcomings of existing technologies and solve the aforementioned problems, this invention provides a method for preparing collagen-based toothpaste raw materials for oral use and its application. This invention is based on recombinant collagen and hydroxyapatite, and focuses on the combined use of aqueous and oil-based solvents, which enables Hap and collagen to better form polymers. At the same time, the combined use of collagen, NHS, and EDC can effectively prevent bacterial invasion, enhance antibacterial performance, and make the anti-caries function more prominent and complete.

[0010] On the other hand, dentin hypersensitivity occurs due to the exposure of enamel and dentin, causing external stimuli to cause the contents of the dentinal tubules to flow outwards, thereby stimulating the dental pulp nerve and producing pain. The final product of this invention is in powder form (nanoscale), with a fine texture and delicate structure, making it more suitable for oral application. It can effectively seal the dentinal tubules, reducing their permeability and thus inhibiting the symptoms of tooth sensitivity and pain. Collagen is required in the enamel repair process, and the added recombinant collagen can accelerate this process; furthermore, the recombinant collagen acts as an adhesive, bonding the easily dispersed nanoscale hydroxyapatite to the enamel, increasing repair efficiency. This invention has undergone substantial research from raw materials, dosage control, and process to the final product form. The resulting product is beneficial for oral application, enhancing the remineralization and desensitizing effects of hydroxyapatite, achieving a combination of gingival protection, caries prevention, and anti-inflammatory functions.

[0011] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution;

[0012] A method for preparing a collagen-based toothpaste raw material for oral use includes the following steps:

[0013] (1) Dissolve the recombinant collagen raw material in water for injection and stir at a temperature not exceeding 40°C to form a collagen solution;

[0014] (2) Preparation of hydroxyapatite suspension: Add hydroxyapatite and aqueous solvent to the oil phase solvent and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain hydroxyapatite suspension; the hydroxyapatite is nano-hydroxyapatite; the ratio of the amount of oil phase solvent, nano-hydroxyapatite and aqueous solvent is 50-100mL: 5-10g: 0.125g.

[0015] (3) Add EDC to water for injection to obtain EDC solution; then add NHS to water for injection to obtain NHS solution; then mix EDC solution and NHS solution and shake evenly to obtain a mixture; then add the mixture to the collagen solution obtained in step (1) to obtain collagen mixture.

[0016] (4) Add the hydroxyapatite suspension obtained in step (2) to the collagen mixture in step (3), wherein the ratio of the amount of hydroxyapatite in the hydroxyapatite suspension to the amount of recombinant collagen in the collagen solution is 5-10g: 0.5-5g; after ultrasonic oscillation until fully mixed; finally, centrifuge, discard the supernatant, collect the precipitate, and air dry it naturally to obtain the raw material for oral collagen-based toothpaste.

[0017] The recombinant collagen mentioned in step (1) includes: recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen.

[0018] In step (1), the ratio of recombinant collagen raw material to water for injection is 0.5-5g:1L; and the stirring and dissolving temperature is 20-40℃.

[0019] The aqueous solvent in step (2) includes: carbomer, xanthan gum, magnesium aluminum silicate, water-soluble cellulose, and water for injection;

[0020] The oil phase solvent mentioned in step (2) includes: vegetable oil, synthetic oil, glycerides, fatty alcohols and beeswax.

[0021] More preferably, the aqueous solvent is a carbomer homopolymer (type C); and the oil solvent is a vegetable oil.

[0022] In step (3), the ratio of EDC to water is 0.2-0.4 mg:1 mL, and the ratio of NHS to water is 0.04-0.08 mg:1 mL; the volume ratio of EDC solution to NHS solution is 1:1.

[0023] In step (3), the ratio of the amount of EDC and NHS in the mixed solution to the amount of recombinant collagen in the collagen solution is 0.2-0.4 mg: 0.04-0.08 mg: 0.5-5 g.

[0024] In step (4), the temperature conditions for ultrasonic oscillation are 25-30℃; the centrifugation conditions are 7200 rpm and centrifugation at 4℃ for 10-15 min.

[0025] The collagen-based toothpaste raw material prepared based on the above steps is used to prepare oral care products for preventing tooth decay and reducing dentin hypersensitivity.

[0026] The oral care products include medications or toothpaste.

[0027] The beneficial effects of this invention are as follows:

[0028] This invention provides a method for preparing collagen-based toothpaste raw materials for oral use and its application; based on recombinant collagen and hydroxyapatite, the key is to use a combination of aqueous and oil-based solvents, which enables hydroxyapatite and collagen to better form polymers; moreover, the selection of their dosage is also crucial, and the polymer can be formed within a limited dosage range, otherwise it will appear as a sponge rather than a powder, and the stability of the prepared raw materials will be reduced.

[0029] Meanwhile, the combination of recombinant collagen, NHS and EDC in this invention can effectively prevent bacterial invasion, and has good gingival protection and anti-inflammatory functions. The combination of specific materials makes the anti-caries function more prominent and complete, and can enhance the remineralization and desensitization effects of hydroxyapatite.

[0030] On the other hand, dentin hypersensitivity occurs due to the exposure of enamel and dentin, causing external stimuli to cause the contents of the dentinal tubules to flow outwards, thereby stimulating the dental pulp nerve and producing pain. The collagen-based toothpaste raw material prepared in this invention has the characteristics of fine texture, very small particle size (D50 < 25 μm), and stable structure, which allows it to better penetrate into the dentinal tubules and effectively seal them (dental tubule sealing rate is above 84%), reducing the permeability of the dentinal tubules and thus inhibiting the symptoms of tooth sensitivity and pain.

[0031] Finally, the involvement of collagen in the enamel repair process significantly enhances the repair effect. Furthermore, the recombinant collagen acts as an adhesive, bonding the easily dispersed nano-sized hydroxyapatite to the enamel and increasing the repair efficiency. Attached Figure Description

[0032] Figure 1 The image shows a product of the collagen-based toothpaste raw material for oral use prepared in Example 1.

[0033] Figure 2 SEM image of the collagen-based toothpaste raw material for oral use prepared in Example 1.

[0034] Figure 3 This is a diagram of the basic structure of a tooth.

[0035] Figure 4 The images show SEM images of different treatment groups in the caries prevention test in Example 1, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-I.

[0036] Figure 5 The images show SEM images of different treatment groups in the dentin hypersensitivity test in Example 1, where A is the control group, B is hydroxyapatite, and C is Hap-Col-I.

[0037] Figure 6 The images show SEM images of different treatment groups in the caries prevention test in Example 2, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-III.

[0038] Figure 7 The images show SEM images of different treatment groups in the dentin hypersensitivity test in Example 2, where A is the control group, B is hydroxyapatite, and C is Hap-Col-III.

[0039] Figure 8 The images show SEM images of different treatment groups in the caries prevention test in Example 3, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-XVII.

[0040] Figure 9The images show SEM images of different treatment groups in the dentin hypersensitivity test in Example 3, where A is the control group, B is hydroxyapatite, and C is Hap-Col-XVII. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, which will be apparent to those skilled in the art. Other embodiments derived from this specification will be obvious to those skilled in the art; the specification and embodiments of this invention are merely exemplary, such as the collagen provided being recombinant human type III collagen; however, this invention is not limited to the selected collagen, and collagen with the same or similar effects is considered a conventional substitution for this invention.

[0045] This invention first provides a raw material for oral collagen-based toothpaste, comprising:

[0046] (1) Raw material selection:

[0047] a. Recombinant collagen (purchased from Jiangsu Chuangjian Medical Technology Co., Ltd.);

[0048] b. Nano-sized hydroxyapatite;

[0049] c. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS RN: 25952-53-8, EDC), N-hydroxysuccinimide (CAS RN: 6066-82-6, NHS);

[0050] d. Aqueous solvent: Carbomer homopolymer (type C) is selected (any water-soluble polymer rheology modifier can be used as an aqueous solvent, such as xanthan gum, carbomer, magnesium aluminum silicate, water-soluble cellulose, etc.);

[0051] e. Oil phase solvent: Vegetable oil (vegetable oil, synthetic oil, glycerides, fatty alcohols, and beeswax are all acceptable);

[0052] Raw material description: The reagents and raw materials used in this invention are all existing products. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) are available from Tokyo Chemical Industry Co., Ltd. (TCI). The recombinant collagen used is collagen obtained through genetic engineering. Carbomer homopolymer (type C) was purchased from Guangzhou Kangqiao Hanpu Pharmaceutical Co., Ltd., and the vegetable oil is available from common supermarkets.

[0053] (2) Preparation method of collagen-based toothpaste raw materials:

[0054] I. Preparation of collagen solution: Add recombinant collagen sponge to injection water and stir until the collagen sponge is fully dissolved and there are no visible suspended matter to obtain collagen solution;

[0055] II. Preparation of hydroxyapatite suspension: Add nano-sized hydroxyapatite and carbomer homopolymer (type C) to vegetable oil and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain a hydroxyapatite suspension.

[0056] III. Preparation of NHS and EDC solutions: Add EDC and NHS to the injection water, shake the two solutions thoroughly and mix them to obtain a mixture; then add the mixture to the collagen solution to obtain a collagen mixture.

[0057] IV. Place the hydroxyapatite suspension in the above collagen solution into an ultrasonic vibrator and ultrasonically vibrate until fully mixed;

[0058] V. Centrifuge the mixed solution, discard the supernatant, collect the precipitate, and air dry it to obtain the target raw material.

[0059] Group experiment:

[0060] The preparation method of collagen-based toothpaste raw materials is the same as in Example 1 below, except that the corresponding reagents (collagen, hydroxyapatite, vegetable oil, carbomer, EDC, NHS) are added according to Table 1, and the results of different addition amounts are shown in Table 1.

[0061] Table 1. Protein content of collagen-based toothpaste raw materials at different collagen concentrations.

[0062]

[0063]

[0064] Note: " / " indicates that the raw material failed to form or that collagen content was not detected.

[0065] Table 1 illustrates that for collagen-based toothpaste ingredients formulated with type I, III, and XVII collagen, within a certain collagen concentration range, the protein content increases significantly with increasing collagen dosage. However, keeping the collagen dosage constant while altering the amounts of vegetable oil EDC and NHS has no significant impact on protein content. Keeping the ratio of vegetable oil to carbomer, EDC, and NHS constant while reducing the amount of collagen (e.g., to 0.4) still affects the final product's form.

[0066] Moreover, the present invention focuses on the combined use of aqueous and oil-phase solvents (with a ratio of 0.125:50-100), which enables Hap and collagen to better form polymers. The amount of each solvent is also crucial, and the polymer can only be formed within the specified range. If the amounts of aqueous and oil-phase solvents are changed, the final product will be sponge-like instead of powder, as shown in Table 1. Reducing or increasing the amount of carbomer will directly affect the morphology of the product and reduce the stability of the raw materials.

[0067] Meanwhile, the process conditions of this invention are also crucial. For example, the mixing of Hap and carbomer can ensure that Hap is uniformly dispersed in the solvent; and a stable ratio of EDC and NHS can play a cross-linking role, making the structure of Hap and collagen more compact and stable; resulting in a final product with a fine texture, a very small particle size (D50 < 25 μm), and a stable structure.

[0068] Example 1:

[0069] (1) Raw material selection:

[0070] a. Recombinant type I collagen;

[0071] b. Nano-sized hydroxyapatite;

[0072] c. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS RN: 25952-53-8, EDC), N-hydroxysuccinimide (CAS RN: 6066-82-6, NHS);

[0073] d. Aqueous solvent: Carbomer homopolymer (type C) is selected;

[0074] e. Oil phase solvent: vegetable oil;

[0075] (2) Preparation method of collagen-based toothpaste raw materials:

[0076] I. Preparation of collagen solution: Add 0.5g of recombinant type I collagen sponge to 1000mL of injection water, stir at a temperature below 40℃ until the collagen sponge is fully dissolved and there are no visible suspended matter, thus forming a collagen solution;

[0077] II. Preparation of hydroxyapatite suspension: Add 10g of nano-sized hydroxyapatite and 0.125g of carbomer homopolymer (type C) to 100mL of vegetable oil, and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain a hydroxyapatite suspension.

[0078] III. Preparation of NHS solution and EDC solution: Add 0.2 mg EDC to 1 mL of injection water and 0.04 mg NHS to 1 mL of injection water. Shake the two solutions thoroughly and mix them. Then add them to the collagen solution obtained in step (1) to obtain a collagen mixture.

[0079] IV. Add hydroxyapatite suspension to the collagen mixture obtained in III above, place it in an ultrasonic oscillator, and ultrasonically oscillate at 25°C until fully mixed.

[0080] V. Centrifuge the well-mixed solution at 7200 rpm and 4℃ for 15 min, discard the supernatant, and air dry at 25℃ to obtain the target raw material Hap-Col-I.

[0081] like Figure 1 As shown, Hap-Col-I is a powder with a D50 < 25 μm; Figure 2 Specifically, the SEM images of the raw material for oral collagen-based toothpaste show the following particle size data: D10 = 2.322 μm, D50 = 23.28 μm, and D90 = 189 μm. It can be seen that it is nanoscale with a fine structure, which is more conducive to tooth repair.

[0082] (3) Caries prevention test

[0083] Table 2 Test Groups

[0084]

[0085] Operating steps:

[0086] S1. Select fresh calf anterior teeth (basic tooth structure as shown in the image) Figure 3 As shown), the root tissue was cut and removed, and after ultrasonic cleaning, it was stored in PBS solution containing double antibodies; teeth with no cracks, caries, or stains on the crown surface were screened, and after ultrasonic cleaning and polishing, enamel samples were obtained for later use.

[0087] S2, Demineralization: The enamel samples were randomly divided into three groups, including a negative control (NC) and sample groups (sample A and sample B). A 0.1%-0.3% lactic acid solution was prepared. The negative control (NC) and sample group samples were immersed in the 0.1%-0.3% lactic acid solution for 3-5 minutes each time, 3-4 times a day, for a total of 2-3 weeks. During the intermittent period, they were placed in artificial saliva at 25-30℃.

[0088] Remineralization: Take samples A and B, add hydroxyapatite and Hap-Col-I respectively, and brush them with teeth. The negative control is also brushed with teeth, but no substances are added. After treatment, the sample group and the negative control group are soaked in 2-3 mL of artificial saliva. The operation is repeated 3 times a day for a total of 3-5 weeks.

[0089] S3. Each group of samples was cut into enamel cubes with a volume of 2mm × 2mm × 1mm using a physical cutting machine. After ultrasonic cleaning, the cubes were dried at room temperature. After gold sputtering in a vacuum, the changes in surface porosity and enamel pillars were observed under a scanning electron microscope.

[0090] Figure 4 The images show SEM images of different treatment groups in the caries prevention test of Example 1, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-I. The SEM images show that after demineralization treatment, the NC group exhibits a loose arrangement of enamel particles, with numerous micropores and an uneven honeycomb-like and fibrous structure, indicating successful modeling in this test. After hydroxyapatite remineralization treatment (4 weeks), the arrangement and structure of enamel particles improved, while after Hap-Col-I remineralization, the arrangement of enamel particles became more orderly and dense, and the micropores and uneven honeycomb-like and fibrous structure were significantly improved.

[0091] (4) Dentin hypersensitivity test

[0092] Table 3 Test Groups

[0093]

[0094] Operating steps:

[0095] S1. Dentin sections (fresh calf anterior teeth) were divided into three groups: control group, sample C group, and sample D group. The sections were immersed in a 20%-30% phosphoric acid solution and incubated on a shaker for 10-15 minutes. After immersion, the sections were quickly rinsed with running water for 3-5 minutes. After rinsing, the sections were immersed in deionized water and ultrasonically cleaned for 5-10 minutes to obtain acid-etched sections.

[0096] S2. Preparation before brushing: Immerse the acid-etched sections from step (1) in artificial saliva. Except for brushing time, keep them immersed in artificial saliva and place them in a 25-30℃ incubator. When brushing is required, remove the control group, sample C group, and sample D group, wipe the artificial saliva off the tooth surface, and then brush. For sample C group, weigh 5-10 mg of hydroxyapatite onto the surface of the section, brush directly with a dry toothbrush for 5-10 seconds, then add 50-100 μL of artificial saliva to the surface of the section using a 100 μL pipette, and continue brushing for 5-15 seconds, for a total of 2-3 minutes. The same procedure applies to sample D group, except that hydroxyapatite is replaced with Hap-Col-I. The same brushing procedure applies to the control group, except that hydroxyapatite and Hap-Col-I are not added. After brushing, rinse the surface of the section with water to remove any foam.

[0097] S3. After the sections are dried, perform gold sputtering; observe the dentinal tubule occlusion using SEM and take photos for record-keeping.

[0098] Figure 5 SEM images of different treatment groups in the dentin hypersensitivity test in Example 1 are shown, where A is the control group, B is hydroxyapatite, and C is Hap-Col-I. After phosphate demineralization treatment, the dentinal tubules in the control group were completely exposed, indicating successful modeling. Compared with the control group, the dentinal tubules in the sample group were partially blocked after hydroxyapatite treatment; after brushing with Hap-Col-I, the dentinal tubules in the sample group were completely blocked, and a mineral shell formed on the surface, showing significant effects.

[0099] Table 4 Summary of Dental Tubule Occlusion Rate Data

[0100]

[0101] Compared with the control group, the tubule occlusion rate of the hydroxyapatite sample increased to 62.89%; the tubule occlusion rate of the Hap-Col-I sample increased significantly to 88.14%, indicating that the Hap-Col-I sample can improve the tubule occlusion rate and has the effect of resisting dentin hypersensitivity.

[0102] Example 2:

[0103] (1) Raw material selection:

[0104] a. Recombinant type III collagen;

[0105] b. Nano-sized hydroxyapatite;

[0106] c. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS RN: 25952-53-8, EDC), N-hydroxysuccinimide (CAS RN: 6066-82-6, NHS);

[0107] d. Aqueous solvent: Carbomer homopolymer (type C) is selected;

[0108] e. Oil phase solvent: vegetable oil;

[0109] (2) Preparation method of collagen-based toothpaste raw materials:

[0110] I. Preparation of collagen solution: Add 0.5g of recombinant type III collagen to 1000mL of injection water and stir at a temperature below 40℃ until the collagen is fully dissolved and there are no visible suspended matter to form a collagen solution;

[0111] II. Preparation of hydroxyapatite suspension: Add 10g of nano-sized hydroxyapatite and 0.125g of carbomer homopolymer (type C) to 100mL of vegetable oil, and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain a hydroxyapatite suspension.

[0112] III. Preparation of NHS solution and EDC solution: Add 0.2 mg EDC to 1 mL of injection water and 0.04 mg NHS to 1 mL of injection water. Shake the two solutions thoroughly and mix them. Then add them to the collagen solution obtained in step (1) to obtain a collagen mixture.

[0113] IV. Add hydroxyapatite suspension to the collagen mixture obtained in step III above, place it in an ultrasonic oscillator, and ultrasonically oscillate at 25°C until fully mixed.

[0114] V. Centrifuge the well-mixed solution at 7200 rpm and 4℃ for 15 min, discard the supernatant, and air dry at 25℃ to obtain the target raw material Hap-Col-III.

[0115] (3) Caries prevention test;

[0116] Table 5 Test Groups

[0117]

[0118] Operating steps:

[0119] S1. Select fresh calf anterior teeth, cut and remove the root tissue, clean them with ultrasound, and store them in PBS solution containing double antibodies; screen teeth with no cracks, caries, or stains on the crown surface, clean them with ultrasonic vibration, polish them, and obtain enamel samples for later use.

[0120] S2. Demineralization: Enamel samples were randomly divided into three groups: negative control (NC), sample group (sample A and sample B). A 0.1%-0.3% lactic acid solution was prepared, and the negative control (NC) and sample group samples were immersed in the 0.1%-0.3% lactic acid solution for 3-5 minutes each time, 3-4 times a day, for a total of 2-3 weeks. During the intermittent period, they were placed in artificial saliva at 25-30℃.

[0121] Remineralization: Take samples A and B, add hydroxyapatite and Hap-Col-III respectively, and brush them with teeth. The negative control is also brushed with teeth, but no substances are added. After treatment, the sample group and the negative control group are soaked in 2-3 mL of artificial saliva. The operation is repeated 3-5 times a day for a total of 2-3 weeks.

[0122] S3. Each group of samples was cut into enamel cubes with a volume of 2mm × 2mm × 1mm using a physical cutting machine. After ultrasonic cleaning, the cubes were dried at room temperature. After gold sputtering in a vacuum, the changes in surface porosity and enamel pillars were observed under a scanning electron microscope.

[0123] Figure 6 The images show SEM images of different treatment groups in the caries prevention test of Example 2, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-III. The SEM images show that after demineralization, the NC group exhibits a loose arrangement of enamel particles with numerous micropores and an uneven honeycomb-like and fibrous structure, indicating successful modeling in this test. Four weeks of hydroxyapatite remineralization improved the arrangement and structure of enamel particles. After Hap-Col-III remineralization, the enamel particles became more orderly and dense, and the micropores and uneven honeycomb-like and fibrous structure were significantly improved.

[0124] (4) Dentin hypersensitivity test

[0125] Table 6 Test Groups

[0126]

[0127] Operating steps:

[0128] S1. Dentin sections (fresh calf anterior teeth) were divided into three groups: control group, sample C group, and sample D group. The sections were immersed in a 20%-30% phosphoric acid solution and incubated on a shaker for 10-15 minutes. After immersion, the sections were quickly rinsed with running water for 3-5 minutes. After rinsing, the sections were immersed in deionized water and ultrasonically cleaned for 5-10 minutes to obtain acid-etched sections.

[0129] S2. Preparation before brushing: Immerse the acid-etched sections from step (1) in artificial saliva. Except for the brushing time, keep them immersed in artificial saliva and place them in a 25-30℃ incubator for constant temperature. When brushing is required, remove the control group, sample C group, and sample D group, wipe the artificial saliva off the tooth surface, and then brush. For sample C group, weigh 5-10 mg of hydroxyapatite onto the surface of the section, brush directly with a dry toothbrush for 5-10 seconds, then add 50-100 μL of artificial saliva to the surface of the section using a 100 μL pipette, and continue brushing for 5-15 seconds, for a total of 2-3 minutes. The same procedure applies to sample D group, except that hydroxyapatite is replaced with Hap-Col-III. The same brushing procedure applies to the control group, except that hydroxyapatite and Hap-Col-III are not added. After brushing, rinse the surface of the section with clean water to remove any foam.

[0130] S3. After the sections are dried, perform gold sputtering; observe the dentinal tubule occlusion using SEM and take photos for record-keeping.

[0131] Figure 7 The images show SEM images of different treatment groups in the dentin hypersensitivity test in Example 2, where A is the control group, B is hydroxyapatite, and C is Hap-Col-III.

[0132] After phosphate demineralization treatment, the dentin sections of the test model showed complete exposure of the dentinal tubules in the control group, indicating successful model establishment. Compared to the BC group, the dentinal tubules in the sample group were partially blocked after treatment with hydroxyapatite. After brushing with Hap-Col-III, the dentinal tubules in the sample group were completely blocked, and a mineral shell formed on the surface, demonstrating significant effectiveness.

[0133] Table 7 Summary of Dental Tubule Occlusion Rate Data

[0134]

[0135] Compared with the control group, the tubule occlusion rate of the hydroxyapatite sample increased to 62.43%. The tubule occlusion rate of the Hap-Col-III sample increased significantly to 85.63%, indicating that the Hap-Col-III sample can improve the tubule occlusion rate and has an anti-dentin hypersensitivity effect.

[0136] Example 3:

[0137] (1) Raw material selection:

[0138] a. Recombinant XVII collagen;

[0139] b. Nano-sized hydroxyapatite;

[0140] c. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS RN: 25952-53-8, EDC), N-hydroxysuccinimide (CAS RN: 6066-82-6, NHS);

[0141] d. Aqueous solvent: Carbomer homopolymer (type C) is selected;

[0142] e. Oil phase solvent: vegetable oil;

[0143] (2) Preparation method of collagen-based toothpaste raw materials:

[0144] I. Preparation of collagen solution: Add 0.5g of recombinant XVII collagen to 1000mL of injection water and stir at a temperature below 40℃ until the collagen is fully dissolved and there are no visible suspended matter to form a collagen solution;

[0145] II. Preparation of hydroxyapatite suspension: Add 10g of nano-sized hydroxyapatite and 0.125g of carbomer homopolymer (type C) to 100mL of vegetable oil, and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain a hydroxyapatite suspension.

[0146] III. Preparation of NHS solution and EDC solution: Add 0.2 mg EDC to 1 mL of injection water and 0.04 mg NHS to 1 mL of injection water. Shake the two solutions thoroughly and mix them. Then add them to the collagen solution obtained in step (1) to obtain a collagen mixture.

[0147] IV. Add hydroxyapatite suspension to the collagen mixture obtained in step III above, place it in an ultrasonic oscillator, and ultrasonically oscillate at 25°C until fully mixed.

[0148] V. Centrifuge the well-mixed solution at 7200 rpm and 4℃ for 15 min, discard the supernatant, and air dry at 25℃ to obtain the target raw material Hap-Col-XVII.

[0149] (3) Caries prevention test

[0150] Table 8 Test Groups

[0151]

[0152] Operating steps:

[0153] S1. Select fresh calf anterior teeth, cut and remove the root tissue, clean them with ultrasound, and store them in PBS solution containing double antibodies; screen teeth with no cracks, caries, or stains on the crown surface, clean them with ultrasonic vibration, polish them, and obtain enamel samples for later use.

[0154] S2, Demineralization: The enamel samples were randomly divided into three groups, including a negative control (NC) and sample groups (sample A and sample B). A 0.1%-0.3% lactic acid solution was prepared. The negative control (NC) and sample group samples were immersed in the 0.1%-0.3% lactic acid solution for 3-5 minutes each time, 3-4 times a day, for a total of 2-3 weeks. During the intermittent period, they were placed in artificial saliva at 25-30℃.

[0155] Remineralization: Take samples A and B, add hydroxyapatite and Hap-Col-XVII respectively, and brush them with teeth. The negative control is also brushed with teeth, but no substances are added. After treatment, the sample group and the negative control group are soaked in 2-3 mL of artificial saliva. The operation is repeated 3-5 times a day for a total of 2-3 weeks.

[0156] S3. Each group of samples was cut into enamel cubes with a volume of 2mm × 2mm × 1mm using a physical cutting machine. After ultrasonic cleaning, the cubes were dried at room temperature. After gold sputtering in a vacuum, the changes in surface porosity and enamel pillars were observed under a scanning electron microscope.

[0157] Figure 8 The images show SEM images of different treatment groups in the caries prevention test in Example 3, where A is the negative control, B is hydroxyapatite, and C is Hap-Col-XVII.

[0158] The SEM images show that after demineralization, the NC group exhibits a loosely arranged microparticle structure on the enamel surface, with numerous micropores and an uneven honeycomb-like and fibrous structure, indicating successful modeling in this test. Four weeks of hydroxyapatite remineralization treatment improved the microparticle arrangement and structure of the enamel surface. After Hap-Col-XVII remineralization, the microparticle arrangement on the enamel surface became more orderly and dense, and the micropores and uneven honeycomb-like and fibrous structures were significantly improved.

[0159] (4) Dentin hypersensitivity test:

[0160] Table 9 Test Groups

[0161]

[0162]

[0163] Operating steps:

[0164] S1. Dentin sections (fresh calf anterior teeth) were divided into three groups: control group, sample C group, and sample D group. The sections were immersed in a 20%-30% phosphoric acid solution and incubated on a shaker for 10-15 minutes. After immersion, the sections were quickly rinsed with running water for 3-5 minutes. After rinsing, the sections were immersed in deionized water and ultrasonically cleaned for 5-10 minutes to obtain acid-etched sections.

[0165] S2. Preparation before brushing: Immerse the acid-etched sections from step (1) in artificial saliva. Except for the brushing time, keep them immersed in artificial saliva and place them in a 25-30℃ incubator for constant temperature. When brushing is required, remove the control group, sample C group, and sample D group, wipe the artificial saliva off the tooth surface, and then brush. For sample C group, weigh 5-10 mg of hydroxyapatite onto the surface of the section, brush directly with a dry toothbrush for 5-10 seconds, then add 50-100 μL of artificial saliva to the surface of the section using a 100 μL pipette, and continue brushing for 5-15 seconds, for a total of 2-3 minutes. The same procedure applies to sample D group, except that hydroxyapatite is replaced with Hap-Col-XVII. The same brushing procedure applies to the control group, except that hydroxyapatite and Hap-Col-XVII are not added. After brushing, rinse the surface of the section with clean water to remove any foam.

[0166] S3. After the sections are dried, perform gold sputtering; observe the dentinal tubule occlusion using SEM and take photos for record-keeping.

[0167] Figure 9 The images show SEM images of different treatment groups in the dentin hypersensitivity test in Example 3, where A is the control group, B is hydroxyapatite, and C is Hap-Col-XVII.

[0168] After demineralization with phosphate, the dentin sections of the test model showed complete exposure of the dentinal tubules in the control group, indicating successful model establishment. Compared to the control group, the dentinal tubules in the sample group were partially blocked after treatment with hydroxyapatite. After brushing with Hap-Col-XVII, the dentinal tubules in the sample group were completely blocked, and a mineral shell formed on the surface, demonstrating significant effectiveness.

[0169] Table 10 Summary of Dental Tubule Occlusion Rate Data

[0170]

[0171] Compared with the control group, the tubule occlusion rate of the hydroxyapatite sample increased to 62.12%. The tubule occlusion rate of the Hap-Col-XVII sample increased significantly to 84.26%, indicating that the Hap-Col-XVII sample can improve the tubule occlusion rate and has an anti-dentin hypersensitivity effect.

[0172] In summary, by using different types of recombinant collagen combined with the raw materials of this invention, and by combining aqueous and oil solvents and controlling the dosage, the prepared oral collagen-based toothpaste raw materials all have the effects of preventing caries and reducing dentin hypersensitivity, and the tubule sealing rate is all above 84%, achieving unexpected and significant results.

[0173] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a collagen-based toothpaste raw material for oral use, characterized in that, The steps are as follows: (1) Dissolve the recombinant collagen raw material in water for injection and stir at a temperature not exceeding 40°C to form a collagen solution; (2) Preparation of hydroxyapatite suspension: Add hydroxyapatite and aqueous solvent to the oil phase solvent, and stir thoroughly until the hydroxyapatite is uniformly dispersed in the solution to obtain a hydroxyapatite suspension; the hydroxyapatite is nano-hydroxyapatite; the ratio of the amount of oil phase solvent, nano-hydroxyapatite and aqueous solvent is 50~100mL: 5-10g: 0.125g; the aqueous solvent is carbomer homopolymer C type; the oil phase solvent is vegetable oil; (3) Add EDC to water for injection to obtain EDC solution; then add NHS to water for injection to obtain NHS solution, then mix EDC solution and NHS solution and shake to obtain mixed solution; The mixed solution was then added to the collagen solution obtained in step (1) to obtain a collagen mixture; the ratio of EDC and NHS in the mixed solution to recombinant collagen in the collagen solution was 0.2-0.4 mg: 0.04-0.08 mg: 0.5-5 g. (4) Add the hydroxyapatite suspension obtained in step (2) to the collagen mixture in step (3), wherein the ratio of hydroxyapatite in the hydroxyapatite suspension to recombinant collagen in the collagen solution is 5-10g: 0.5-5g; after ultrasonic oscillation until fully mixed; finally, centrifuge, discard the supernatant, collect the precipitate, and air dry it naturally to obtain the raw material for oral collagen-based toothpaste.

2. The method for preparing a collagen-based toothpaste raw material for oral use according to claim 1, characterized in that, The recombinant collagen mentioned in step (1) includes: recombinant type I collagen, recombinant type III collagen, and recombinant type XVII collagen.

3. The method for preparing a collagen-based toothpaste raw material for oral use according to claim 1, characterized in that, The ratio of recombinant collagen raw material to water for injection in step (1) is 0.5-5g:1L; the stirring and dissolving temperature is 20-40℃.

4. The method for preparing a collagen-based toothpaste raw material for oral use according to claim 1, characterized in that, In step (4), the temperature conditions for ultrasonic oscillation are 25~30℃; the centrifugation conditions are: 7200rpm, centrifugation at 4℃ for 10~15min.

5. The oral collagen-based toothpaste raw material prepared according to any one of claims 1 to 4, characterized in that, The raw material for the oral collagen-based toothpaste is in powder form with a D50 < 25 μm.

6. The use of the oral collagen-based toothpaste raw material according to claim 5 in the preparation of oral care products for preventing caries and reducing dentin hypersensitivity.

7. The use according to claim 6, characterized in that, The oral care products include medications or toothpaste.

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