Polypeptide and application thereof in preparation of product for inhibiting tooth demineralization

Abstract

The invention provides a polypeptide and application thereof in preparation of a product for inhibiting tooth demineralization. The polypeptide with the amino acid sequence shown as SEQ ID NO: 2 can effectively adjust the pH value of the oral cavity, further remarkably inhibits tooth demineralization and is suitable for preparing products such as drugs for inhibiting tooth demineralization, oral health care products and oral care products. In addition, the polypeptide sequence is short, the production cost is low, and the polypeptide is suitable for batch popularization and application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceutical manufacturing. More particularly, it relates to a polypeptide and its use in preparing a product for inhibiting tooth demineralization. BACKGROUND

[0002] Enamel is the hardest protective layer of the tooth, and its main component is hydroxyapatite. When acidic substances in the oral cavity (such as acids in carbonated beverages, acids produced by bacterial metabolism) continuously act, the pH value of the oral cavity will decrease significantly, causing the dissolution and loss of calcium, phosphorus and other minerals in the enamel, resulting in tooth demineralization.

[0003] Tooth demineralization can cause many adverse effects on the human body, such as: (1) periodontal tissue involvement: the surface of demineralized teeth is rough, easy to adsorb dental plaque, increasing the risk of periodontitis and gingivitis, manifested as redness, gum bleeding, tooth loosening, etc.; (2) tooth sensitivity: demineralization makes the enamel thin, exposing the dentin, and causing pain to cold, heat, acid and sweet stimuli, and daily eating (such as eating sweets, drinking ice water) may cause transient but severe pain; (3) abnormal occlusion function: severe demineralization may cause changes in tooth shape, affecting the occlusal relationship, and causing temporomandibular joint disorders (such as joint clicking, pain, etc.), and decreased masticatory efficiency; (4) damage to appearance: the tooth surface appears chalky or yellow-brown spots, the color is uneven, and severe demineralization may cause the enamel surface to be concave or grooved, further damaging the tooth shape; (5) structural weakness: demineralized teeth have decreased hardness and are more prone to wear, cracking, and even breaking when chewing hard objects; (6) systemic health risks: oral infections can spread to the whole body through the blood, increasing the risk of respiratory diseases, heart disease, diabetes and other chronic diseases; children's tooth demineralization may also affect nutrient intake and maxillofacial development. Therefore, it is urgent to find a product that can inhibit tooth demineralization by adjusting the pH value of the oral cavity.

[0004] Polypeptides are compounds formed by connecting multiple alpha-amino acids together through a peptide chain. Due to their various biological activities and the obvious advantages of not causing adverse reactions and drug resistance, polypeptides have gradually attracted widespread attention in the pharmaceutical field. However, there are still few polypeptides that can be used to inhibit tooth demineralization. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provides a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2, which can effectively regulate the pH value of the oral cavity and significantly inhibit tooth demineralization, and is suitable for preparing products such as drugs, oral health products and oral care products for inhibiting tooth demineralization.

[0006] The first object of the present application is to provide a polypeptide.

[0007] The second object of the present application is to provide related biological materials of the above-mentioned polypeptide.

[0008] A third object of the present application is to provide use of the polypeptide or the related biological material in the preparation of a product for inhibiting tooth demineralization.

[0009] A fourth object of the present application is to provide a pharmaceutical product.

[0010] A fifth object of the present application is to provide an oral care product.

[0011] A sixth object of the present application is to provide an oral care product.

[0012] The above objects of the present application are achieved by the following technical solutions: The polypeptide with the amino acid sequence as shown in SEQ ID NO: 2 can effectively regulate the pH value of the oral cavity, and thus significantly inhibit tooth demineralization, and is suitable for the preparation of a pharmaceutical product, an oral care product, an oral care product, and the like. Therefore, the present application provides: (1) A polypeptide, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2.

[0013] (2) A related biological material of the polypeptide, wherein the related biological material is a nucleic acid molecule capable of encoding the polypeptide, or an expression cassette, a recombinant plasmid, a recombinant bacteria, or a recombinant cell containing the nucleic acid molecule.

[0014] (3) Use of the polypeptide or the related biological material in the preparation of a product for inhibiting tooth demineralization.

[0015] (4) A pharmaceutical product containing the polypeptide or the related biological material.

[0016] (5) An oral care product containing the polypeptide or the related biological material.

[0017] (6) An oral care product containing the polypeptide or the related biological material.

[0018] Preferably, the product is one of a pharmaceutical product, an oral care product, and an oral care product.

[0019] Preferably, the pharmaceutical product is one of an oral liquid, a suppository, a spirit agent, a powder aerosol, an aerosol, a tincture, a sterile powder for injection, and an injection.

[0020] Further preferably, the pharmaceutical product further contains an excipient.

[0021] More preferably, the excipients are vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter, used as solvents or carriers to help the drug components dissolve or disperse better), wetting agents (such as sodium lauryl sulfate, used to help the drug components disperse and dissolve better), binders (such as gelatin, used to help the drug components bind together better), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and / or methyl cellulose, used to improve the compressibility and formability of the drug, and also to control the release rate of the drug), emulsifiers (such as Tween and / or polyoxyethylene castor oil, used to reduce interfacial tension, promote emulsification of the dispersed phase, and maintain the stability of the emulsion), flavoring agents (used to improve the taste of the drug and improve patient compliance), antioxidants (used to prevent the drug from deteriorating due to oxidation during storage or use and extend the shelf life of the drug), sugars (such as lactose, glucose, and / or sucrose, used to help the drug components disperse and dissolve better, while providing necessary energy support), and polyols (such as propylene glycol, glycerin, sorbitol, and mannose). Alcohols and / or polyethylene glycol, etc., used to improve the stability of drugs, prevent drugs from losing water or deteriorating during storage, and also to improve the taste and solubility of drugs; stabilizers (to increase the physical stability of drugs and prevent them from deteriorating or degrading during storage or transportation); solid lubricants (such as stearic acid and / or magnesium stearate, etc., used to reduce friction and ensure successful drug manufacturing); starches (such as corn starch and / or potato starch, etc., used to help tablets disintegrate rapidly after administration and release drug components, thereby improving the bioavailability of drugs); isotonic salt solutions (used to adjust the osmotic pressure of drugs to meet the physiological requirements of the human body, thereby reducing irritation and adverse reactions to the human body); lubricants (such as talc, etc., used to reduce friction of drugs); colorants (used to improve the appearance and color of drugs, making them easier to identify and distinguish); tablet compressors (facilitating the compression of loose granular substances into solid tablets, making it convenient for patients to take and carry, and also helping to improve the stability and bioavailability of drugs); alginic acid (to maintain the physical stability of drugs and prevent drug particles from settling or agglomerating). The type of excipient can be selected based on the need to improve drug stability, activity, and / or bioavailability.

[0022] Preferably, the oral care product is one of the following: oral patch, oral gel, oral lozenge, toothpaste, oral powder, oral care liquid, mouthwash, oral sustained-release agent, mouthwash effervescent tablet, and oral spray.

[0023] More preferably, the oral care product also contains excipients.

[0024] More preferably, the excipients are cooling agents (such as menthol and its derivatives, used to increase the cooling sensation of oral care products and enhance the user's sensory experience), flavorings (such as peppermint flavoring, used to increase the aroma and taste of oral care products and enhance the user's sensory experience), solvents (such as glycerin, propylene glycol and / or ethanol, used to dissolve raw materials for easy use and storage), sweeteners (such as sucralose, sodium saccharin, sorbitol, xylitol and / or maltitol, used to increase the sweetness of oral care products and enhance the user's sensory experience), and antibacterial agents (such as cetylpyridinium chloride, chlorhexidine gluconate and / or benzalkonium chloride, etc.). One or more of the following excipients are used to inhibit or kill harmful microorganisms in the oral cavity, helping to maintain oral health: excipients (such as potassium sorbate, sodium benzoate, methylparaben, and / or propylparaben, used to prevent the growth of microorganisms in oral care products and extend their shelf life), surfactants (such as sodium lauryl sulfate and / or sodium lauroyl sarcosinate, used to reduce the surface tension of oral care products and enhance cleaning effectiveness), and thickeners (such as poloxamer 407, used to increase the viscosity of oral care products, improve the user experience, and also to improve the stability of oral care products, preventing them from separating or settling). The type of excipient can be selected according to the need to improve the stability, safety, effectiveness, or user experience of oral care products.

[0025] Preferably, the oral care product is one of the following: oral patch, oral gel, oral lozenge, toothpaste, oral powder, oral care liquid, mouthwash, oral sustained-release agent, mouthwash effervescent tablet, and oral spray.

[0026] More preferably, the oral care product also contains excipients.

[0027] More preferably, the excipients are cooling agents (such as menthol and its derivatives, used to increase the cooling sensation of oral care products and enhance the user's sensory experience), flavorings (such as peppermint flavoring, used to increase the aroma and taste of oral care products and enhance the user's sensory experience), solvents (such as glycerin, propylene glycol and / or ethanol, used to dissolve raw materials for easy use and storage), sweeteners (such as sucralose, sodium saccharin, sorbitol, xylitol and / or maltitol, used to increase the sweetness of oral care products and enhance the user's sensory experience), and antibacterial agents (such as cetylpyridinium chloride, chlorhexidine gluconate and / or benzalkonium chloride, etc.). One or more of the following excipients are used to inhibit or kill harmful microorganisms in the oral cavity, helping to maintain oral health: (1) excipients; (2) preservatives (such as potassium sorbate, sodium benzoate, methylparaben, and / or propylparaben, used to prevent the growth of microorganisms in oral care products and extend their shelf life); (3) surfactants (such as sodium lauryl sulfate and / or sodium lauroyl sarcosinate, used to reduce the surface tension of oral care products and enhance cleaning effectiveness); and (4) thickeners (such as poloxamer 407, used to increase the viscosity of oral care products, improve the user experience, and also to improve the stability of oral care products, preventing them from separating or settling). The type of excipient can be selected based on the need to improve the stability, safety, effectiveness, or user experience of oral care products.

[0028] The present invention has the following beneficial effects: 1. The polypeptide of the present invention, with an amino acid sequence as shown in SEQ ID NO:2, can effectively regulate oral pH and thus significantly inhibit tooth demineralization. It is suitable for preparing pharmaceuticals, oral health care products, oral care products, and other products that inhibit tooth demineralization.

[0029] 2. The polypeptide sequence of the present invention is relatively short, resulting in lower production costs and making it suitable for mass production and application. Attached Figure Description

[0030] Figure 1 This is the HPLC chromatogram of the control peptide.

[0031] Figure 2 The image shows the HPLC spectrum of the polypeptide obtained in Example 1.

[0032] Figure 3 The image shows the HPLC spectrum of the polypeptide obtained in Comparative Example 1.

[0033] Figure 4 Linear graphs showing the pH values ​​of four groups of saliva over 5 hours.

[0034] Figure 5 A bar chart showing the hourly pH values ​​of four groups of saliva.

[0035] Figure 6The figure shows the test results of the long-term inhibitory effect of saliva on tooth demineralization in four groups. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0038] Example 1 Synthesis of Polypeptides The polypeptide with the amino acid sequence shown in SEQ ID NO:1 was used as the control polypeptide (SEQ ID NO:1: GLDWWQL, synthesized by Sangon Biotech Co., Ltd., HPLC chromatogram as shown). Figure 1 As shown), an L-glutamic acid was inserted before the second amino acid at the N-terminus to obtain the polypeptide of Example 1 (amino acid sequence as shown in SEQ ID NO:2: GELDWWQL, synthesized by Sangon Biotech Co., Ltd., HPLC chromatogram as shown). Figure 2 (As shown).

[0039] Comparative Example 1 Using the polypeptide with the amino acid sequence shown in SEQ ID NO:1 as a control polypeptide (SEQ ID NO:1: GLDWWQL), an L-aspartic acid was inserted before the second amino acid at the N-terminus to obtain the polypeptide of Comparative Example 1 (amino acid sequence shown in SEQ ID NO:3: GDLDWWQL, synthesized by Sangon Biotech Co., Ltd., HPLC chromatogram as shown). Figure 3 (As shown).

[0040] Test Example 1: Immediate Inhibition of Tooth Demineralization by the Peptide I. Preparation of human filtrated saliva Saliva was collected from 3 healthy volunteers, mixed well, and then centrifuged at 4 ℃ and 10000 g for 10 min. The supernatant obtained by centrifugation was collected and filtered with a 0.22 μm filter membrane to remove bacteria, thus obtaining the human in vitro filtered saliva.

[0041] II. Test methods for the immediate inhibitory effect of peptides on tooth demineralization Ten volunteers were selected based on the following criteria: (1) natural dentition; (2) no periodontitis or acute caries; (3) plaque on the buccal and lingual surfaces of molars; (4) no use of antibiotics in the past three months; (5) fasting and abstaining from drinking for 2 hours before collection; (6) not brushing teeth for 24 hours before collection.

[0042] Dental plaque was scraped from the buccal and lingual surfaces of volunteers' molars using a sterile probe and dispersed in 500 μL PBS (pH=7.4). 50 μL of the dispersed plaque was inoculated into 1.5 mL of human saliva filtered from vitro (pH=7.0, adjusted with sodium hydroxide and hydrochloric acid) containing 1 wt% glucose. The peptides obtained in Example 1 and Comparative Example 1, as well as the control peptide, were then added to bring the final peptide concentration to 25 μM (the group without peptides was used as the blank group). The mixture was anaerobically cultured at 37 ℃ and 5% CO2 for 5 h, with the pH value of the saliva measured every 1 h during the process.

[0043] III. Test Results of the Immediate Inhibitory Effect of Peptides on Tooth Demineralization The results are as follows Figures 4-5 As shown, where, Figure 4 The graph shows the statistical line graph of pH values ​​of four groups of saliva over 5 hours. Figure 5 A bar chart showing the hourly pH values ​​of four groups of saliva.

[0044] visible: (1) Compared with the blank group, the pH values ​​of the peptides obtained in Example 1, Comparative Example 1 and the control peptides were significantly increased from the 3h, indicating that the three groups of peptides can play a regulatory role on oral pH value in a short time and achieve immediate inhibition of tooth demineralization. (2) Compared with the control peptide, the pH value of the peptide obtained in Comparative Example 1 showed no significant difference from 1 to 5 h, while the pH value of the peptide obtained in Example 1 showed a significant difference from 3 h onwards, and the degree of difference gradually increased over time. This indicates that not all mutations of the control peptide can significantly enhance its immediate inhibitory effect on tooth demineralization. Rather, it is the insertion of a specific amino acid before the second amino acid at the N-terminus of the control peptide in this application that significantly enhances its immediate inhibitory effect on tooth demineralization.

[0045] Test Example 2: Long-term inhibitory effect of peptides on tooth demineralization I. Preparation of human filtrated saliva Saliva was collected from 3 healthy volunteers, mixed well, and then centrifuged at 4 ℃ and 10000 g for 10 min. The supernatant obtained by centrifugation was collected and filtered with a 0.22 μm filter membrane to remove bacteria, thus obtaining the human in vitro filtered saliva.

[0046] II. Test methods for the long-term inhibitory effect of peptides on tooth demineralization Ten volunteers were selected based on the following criteria: (1) natural dentition; (2) no periodontitis or acute caries; (3) plaque on the buccal and lingual surfaces of molars; (4) no use of antibiotics in the past three months; (5) fasting and abstaining from drinking for 2 hours before collection; (6) not brushing teeth for 24 hours before collection.

[0047] Dental plaque was scraped from the buccal and lingual surfaces of volunteers' molars using a sterile probe and dispersed in 500 μL PBS (pH=7.4). 50 μL of the dispersed plaque was inoculated into 1.5 mL of human in vitro filtered saliva containing 1 wt% sucrose (pH=7.0, adjusted with sodium hydroxide and hydrochloric acid). The peptides obtained in Example 1 and Comparative Example 1, as well as the control peptide, were then added to bring the final peptide concentration to 25 μM (the group without peptides was used as the blank group). After anaerobic culture at 37 ℃ and 5% CO2 for 24 h to form a plaque biofilm, the pH value of the saliva was measured at 24 h.

[0048] III. Test Results of the Long-Term Inhibitory Effect of Peptides on Tooth Demineralization The results are as follows Figure 6 As shown, it can be seen that: (1) Compared with the blank group, the pH values ​​of the peptides obtained in Example 1, Comparative Example 1 and the control peptides at 24 h were significantly increased, indicating that the three groups of peptides can maintain a long-term regulatory effect on oral pH and achieve a long-term inhibitory effect on tooth demineralization. (2) Compared with the control peptide, the pH value of the peptide obtained in Comparative Example 1 at 24 h showed no significant difference, while the pH value of the peptide obtained in Example 1 showed a significant difference. This indicates that not all mutations of the control peptide can significantly enhance its long-term inhibitory effect on tooth demineralization. Rather, it is the insertion of a specific amino acid before the second amino acid at the N-terminus of the control peptide in this application that significantly enhances its long-term inhibitory effect on tooth demineralization.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

2.

2. The biomaterials related to the polypeptide of claim 1, characterized in that, The relevant biological material is a nucleic acid molecule that can encode the polypeptide, or an expression cassette, recombinant plasmid, recombinant bacteria, or recombinant cell containing the nucleic acid molecule.

3. The use of the polypeptide of claim 1 or the related biomaterial of claim 2 in the preparation of products that inhibit tooth demineralization.

4. The application according to claim 3, characterized in that, The product is one of the following: pharmaceuticals, oral health care products, or oral care products.

5. The application according to claim 4, characterized in that, The drug is one of the following: oral liquid, suppository, medicated liquid, powder inhaler, aerosol, tincture, sterile powder for injection, or injection.

6. The application according to claim 4, characterized in that, The oral health care products mentioned are one of the following: oral patches, oral gels, oral lozenges, toothpaste, oral powders, oral care solutions, mouthwash, oral sustained-release agents, mouthwash effervescent tablets, and oral sprays.

7. The application according to claim 4, characterized in that, The oral care products mentioned are one of the following: oral patches, oral gels, oral lozenges, toothpaste, oral powders, oral care liquids, mouthwash, oral sustained-release agents, mouthwash effervescent tablets, and oral sprays.

8. A medicine, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

9. An oral care product, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

10. An oral care product, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

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