Oral care solution and preparation method thereof

Abstract

The invention discloses an oral care solution, which comprises the following components: 1000 ml of deionized water, 2 g of licorice extract, 0.5 ml of tea tree oil, 0.1 g of sodium hyaluronate, a proper amount of citric acid, 0.5 ml of perfume, and food-grade natural mint essence. The oral care solution provided by the invention has obvious antibacterial, anti-inflammatory and moisturizing effects, and can effectively improve oral health. The preparation method is simple and easy to operate, and has high application value. Therefore, the oral care solution prepared by the invention is suitable for large-scale popularization and application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an oral care liquid and a preparation method thereof. Background Art

[0002] Oral health is an important part of overall health, directly affecting the body's digestion, respiration and systemic immune system. In recent years, with the improvement of people's living standards, the demand for oral care products has increased. Oral care solutions on the market are usually used to clean the mouth, eliminate bad breath, prevent gingivitis, etc. However, most of these traditional products focus on antibacterial and cleaning effects, lack protection and repair of oral mucosa, and fail to meet the growing and diverse needs of consumers.

[0003] The health of the oral mucosa is directly related to the comfort and function of the oral cavity. Common oral problems such as oral ulcers, gingivitis and xerostomia are often related to damage, inflammation and dehydration of the oral mucosa. These problems not only affect daily diet, but may also lead to more serious health problems. In addition, the fast pace and high pressure of modern society make many people prone to problems such as oral ulcers and sensitive gums. Therefore, the development of an oral care solution with antibacterial, anti-inflammatory, moisturizing and soothing effects can effectively meet the needs of modern consumers.

[0004] Some oral care solutions currently on the market use natural plant extracts, such as licorice and tea tree oil, which have certain antibacterial and anti-inflammatory effects, but often fail to comprehensively consider the protection of the oral mucosa in the formula. In addition, the alcohol components contained in some products may cause dryness in the mouth, which in turn aggravates certain oral problems. Therefore, it is particularly important to develop a new type of oral care solution that can keep the mouth moist and comfortable while being antibacterial.

[0005] In view of the deficiencies in the prior art, the present invention designs an oral care liquid with multiple functions such as antibacterial, anti-inflammatory and moisturizing by selecting a variety of natural ingredients. The oral care liquid can not only effectively clean the oral cavity and eliminate pathogens, but also protect the oral mucosa in daily use, enhance the user experience, and thus achieve a comprehensive effect of oral care. Summary of the invention

[0006] In order to solve the above technical problems, the present invention discloses an oral care liquid and a preparation method thereof.

[0007] Therefore, the present invention discloses an oral care solution in one aspect, the care solution comprising the following components:

[0008] Deionized water: 1000 ml,

[0009] Licorice extract: 2 g,

[0010] Tea tree oil: 0.5 ml,

[0011] Sodium hyaluronate: 0.1 g,

[0012] Citric acid: appropriate amount,

[0013] Flavor: 0.5 ml food grade natural mint flavor.

[0014] In another aspect, the present invention further discloses an oral care solution, the care solution comprising the following components:

[0015] Deionized water: 1000 ml,

[0016] Licorice extract: 2 g,

[0017] Tea tree oil: 0.5 ml,

[0018] Sodium hyaluronate: 0.1 g,

[0019] Monoclonal antibody against Staphylococcus aureus: 0.1 g,

[0020] Citric acid: appropriate amount,

[0021] Flavor: 0.5 ml food grade natural mint flavor.

[0022] Preferably, the monoclonal antibody against Staphylococcus aureus described in the present invention is an anti-SPA monoclonal antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SPA monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0023] Preferably, the citric acid of the present invention is used to adjust the pH value of the care solution so that the pH value of the care solution is 6.5.

[0024] In another aspect, the present invention further discloses a method for preparing the care solution, the method comprising the following steps:

[0025] (1) Preparation: Clean all instruments, such as stirrers, measuring cylinders, bottles, etc., and ensure they are sterile; operate in a well-ventilated environment;

[0026] (2) Solvent preparation: Pour 1000 ml of deionized water into a blender and turn on the blender at medium speed to ensure uniform water flow;

[0027] (3) Adding licorice extract: Slowly add 2 g of licorice extract into deionized water and stir continuously to ensure that it is completely dissolved. Generally, stir for about 5 minutes.

[0028] (4) Add tea tree oil and sodium hyaluronate: Continue stirring and slowly add 0.5 ml of tea tree oil and 0.1 g of sodium hyaluronate. Keep stirring to ensure uniform dispersion. Generally, stir for about 3 to 5 minutes.

[0029] (5) Addition of monoclonal antibody against Staphylococcus aureus: slowly add 0.1 g of monoclonal antibody against Staphylococcus aureus to ensure uniform mixing;

[0030] (6) Adjusting pH: Gradually add citric acid, use a pH meter to monitor the pH of the solution in real time, and adjust the pH to 6.5 to ensure palatability and safety;

[0031] (7) Adding flavors: Finally, add 0.5 ml of food-grade natural mint flavor and continue stirring for about 5 minutes to mix it thoroughly and ensure that the flavor is evenly distributed;

[0032] (8) Filtration and storage: Aseptically filter the prepared oral care solution through a 0.22 micron sterile filter to remove any microorganisms. Bottle the filtered liquid, seal it, and store it in a cool, dry place away from light.

[0033] In another aspect, the present invention also discloses a use of the anti-SPA monoclonal antibody in preparing an oral care solution.

[0034] The oral care liquid of the present invention has obvious antibacterial, anti-inflammatory and moisturizing effects, and can effectively improve oral health. The preparation method is simple, easy to operate, and has high application value. Therefore, the oral care liquid prepared by the present invention is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the mouse monoclonal antibody preparation process.

[0036] Figure 2 The results of SDS-PAGE detection of anti-SPA monoclonal antibody, where 1 is the marker and 2 is the monoclonal antibody. DETAILED DESCRIPTION

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0038] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] Example 1: Preparation of oral care solution

[0040] 1. Materials

[0041] Deionized water: 1000 ml,

[0042] Licorice extract: 2 g,

[0043] Tea tree oil: 0.5 ml,

[0044] Sodium hyaluronate: 0.1 g,

[0045] Citric acid: appropriate amount (for adjusting pH),

[0046] Flavor: 0.5 ml food grade natural mint flavor.

[0047] 2. Preparation method

[0048] (1) Preparation: Clean all instruments, such as stirrers, measuring cylinders, bottles, etc., and ensure they are sterile; operate in a well-ventilated environment;

[0049] (2) Solvent preparation: Pour 1000 ml of deionized water into a blender and turn on the blender at medium speed to ensure uniform water flow;

[0050] (3) Adding licorice extract: Slowly add 2 g of licorice extract into deionized water and stir continuously to ensure that it is completely dissolved. Generally, stir for about 5 minutes.

[0051] (4) Add tea tree oil and sodium hyaluronate: Continue stirring and slowly add 0.5 ml of tea tree oil and 0.1 g of sodium hyaluronate. Keep stirring to ensure uniform dispersion. Generally, stir for about 3 to 5 minutes.

[0052] (5) Adjusting pH: Gradually add citric acid, use a pH meter to monitor the pH of the solution in real time, and adjust the pH to 6.5 to ensure palatability and safety;

[0053] (6) Adding flavors: Finally, add 0.5 ml of food-grade natural mint flavor and continue stirring for about 5 minutes to mix it thoroughly and ensure that the flavor is evenly distributed;

[0054] (7) Filtration and storage: Aseptically filter the prepared oral care solution through a 0.22 micron sterile filter to remove any microorganisms. Bottle the filtered liquid, seal it, and store it in a cool, dry place away from light.

[0055] Example 2: Preparation of oral care solution

[0056] 1. Materials

[0057] Deionized water: 1000 ml,

[0058] Licorice extract: 2 g,

[0059] Tea tree oil: 0.5 ml,

[0060] Sodium hyaluronate: 0.1 g,

[0061] Monoclonal antibody against Staphylococcus aureus (prepared in Example 4): 0.1 g,

[0062] Citric acid: appropriate amount (for adjusting pH),

[0063] Flavor: 0.5 ml food grade natural mint flavor.

[0064] 2. Preparation method

[0065] (1) Preparation: Clean all instruments, such as stirrers, measuring cylinders, bottles, etc., and ensure they are sterile; operate in a well-ventilated environment;

[0066] (2) Solvent preparation: Pour 1000 ml of deionized water into a blender and turn on the blender at medium speed to ensure uniform water flow;

[0067] (3) Adding licorice extract: Slowly add 2 g of licorice extract into deionized water and stir continuously to ensure that it is completely dissolved. Generally, stir for about 5 minutes.

[0068] (4) Add tea tree oil and sodium hyaluronate: Continue stirring and slowly add 0.5 ml of tea tree oil and 0.1 g of sodium hyaluronate. Keep stirring to ensure uniform dispersion. Generally, stir for about 3 to 5 minutes.

[0069] (5) Addition of monoclonal antibody against Staphylococcus aureus: slowly add 0.1 g of monoclonal antibody against Staphylococcus aureus to ensure uniform mixing;

[0070] (6) Adjusting pH: Gradually add citric acid, use a pH meter to monitor the pH of the solution in real time, and adjust the pH to 6.5 to ensure palatability and safety;

[0071] (7) Adding flavors: Finally, add 0.5 ml of food-grade natural mint flavor and continue stirring for about 5 minutes to mix it thoroughly and ensure that the flavor is evenly distributed;

[0072] (8) Filtration and storage: Aseptically filter the prepared oral care solution through a 0.22 micron sterile filter to remove any microorganisms. Bottle the filtered liquid, seal it, and store it in a cool, dry place away from light.

[0073] Example 3: Application of oral care solution

[0074] 1. Antibacterial experiment: Prepare LB agar plates and inoculate Staphylococcus aureus (ATCC 6538) and oral Streptococcus (ATCC 10241). After streaking the plate, add 10 μL of oral care solution 1 (prepared in Example 1), oral care solution 2 (prepared in Example 2), commercial control (manufacturer: Johnson & Johnson, model: Listerine Cool Mint) and normal saline to the center of the plate. Culture for 24 hours at 37°C. Measure the diameter of the inhibition zone. The results showed that the diameter of the inhibition zone in the experimental group was significantly increased compared with that in the control group (P<0.01), indicating that the oral care solution has a good inhibitory effect on both bacteria. And the effect of oral care solution 2 is better, and oral care solution 1 is equivalent to the commercialized one. The specific results are shown in Table 1.

[0075] Table 1 Inhibition zone diameter test results

[0076]

[0077] 2. Anti-inflammatory experiment: Eight-week-old male mice were used and randomly divided into an experimental group and a control group (10 mice in each group). Oral care solution 1 (prepared in Example 1), oral care solution 2 (prepared in Example 2), a commercial control (manufacturer: Johnson & Johnson, model: Listerine Cool Mint) and normal saline were applied to the ears of the mice. Induce inflammation (using 10% capsaicin solution). The ear thickness was measured after 48 hours. The results showed that the ear thickness of the experimental group was significantly different from that of the control group (P<0.05), indicating that the care solution has a significant anti-inflammatory effect. And the effect of oral care solution 2 is better, and oral care solution 1 is equivalent to or better than the commercial one. The specific results are shown in Table 2.

[0078] Table 2 Ear thickness test results

[0079] Group Ear thickness Oral Care Solution1 1.8mm±0.25mm Oral Care Solution 2 1.3mm±0.21mm Normal saline control group 3.0mm±0.28mm Commercial control group 2.0mm±0.23mm

[0080] 3. Moisturizing experiment: 15 volunteers were recruited and randomly divided into 3 groups: oral care solution 1 (prepared in Example 1), oral care solution 2 (prepared in Example 2) and control group (no care solution). The moisture content of the oral mucosa surface was measured 30 minutes and 1 hour after using the care solution. The results showed that the two groups using the care solution had significantly higher moisture retention than the control group (P<0.01), indicating that the care solution has a good effect in keeping the mouth moist. And the effect of oral care solution 2 is better. The specific results are shown in Table 3.

[0081] Table 3 Moisturizing test results

[0082] Group Moisture content after 30 minutes Moisture content after 1 hour Oral Care Solution1 39％±6％ 33％±5％ Oral Care Solution 2 46％±5％ 42％±4％ Normal saline control group 28％±6％ 23％±5％

[0083] Example 4: Preparation of monoclonal antibodies against Staphylococcus aureus

[0084] 1. Protein A of Staphylococcus aureus is an important surface protein. It is a relatively large membrane-bound protein with multiple immunoglobulin binding domains (IgG binding domains) and is usually bound to the bacterial cell wall. Protein A can bind to the host's immunoglobulins (mainly IgG), especially its Fc part, thereby interfering with the function of the antibody and inhibiting the host's immune response. This allows Staphylococcus aureus to survive and reproduce more effectively in the host. Protein A plays a key role in the formation of bacterial biofilms, helping bacteria to attach and colonize on the surface of host tissues (such as teeth, skin, etc.). Protein A can also trigger local inflammatory responses by activating the host's immune cells (such as macrophages and neutrophils). Although this helps to clear bacteria, it may also cause tissue damage. Therefore, monoclonal antibodies against protein A can be used to neutralize the immune escape of Staphylococcus aureus, enhance the host immune response, and reduce the risk of infection.

[0085] 2. We followed the conventional mouse monoclonal antibody preparation process (such as Figure 1 (as shown) to prepare monoclonal antibodies against protein A of Staphylococcus aureus. A brief description is as follows: Recombinant Staphylococcus aureus protein A (Chengdu Yunxi) is mixed with Freund's complete adjuvant to prepare an immune mixture. The mixture is injected into mice several times to enhance the immune response. After a strong immune response is produced in the mouse, B cells are extracted from its spleen. These B cells are fused with myeloma cells (usually tumor cells that do not produce antibodies) to form hybridoma cells. This step usually uses polyethylene glycol (PEG) as a fusion agent. The fused cell suspension is inoculated into a selection medium containing antibiotics to inhibit the growth of unfused B cells. After screening, the obtained hybridoma cells are transferred to a 96-well plate for monoclonal culture. The production of antibodies is detected by ELISA and other methods. After screening out hybridomas that produce specific antibodies, they are amplified and cultured in appropriate culture media to obtain a large amount of monoclonal antibodies. Then, the obtained antibodies are purified by affinity chromatography or other methods.

[0086] After the above steps, we screened a good monoclonal antibody against Staphylococcus aureus, labeled as anti-SPA monoclonal antibody. After testing, the concentration of the monoclonal antibody was 3.8 mg / ml (BCA test), and the SDS-PAGE purity of the monoclonal antibody was >95% (such as Figure 2The expression level of the monoclonal antibody in ascites can reach 5 mg / ml, so it has a high expression yield, providing a good basis for its large-scale application. At the same time, after sequencing, the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0087] 3. With reference to 7.3 Antibacterial effect test method (suspension quantitative method) of antibacterial daily chemical products under "QBT2738-2012-Evaluation method for antibacterial and antibacterial effects of daily chemical products", the antibacterial activity of anti-SPA monoclonal antibody (active concentration of 0.1g / L) against 5 common pathogenic bacteria was evaluated, and a negative control group (normal saline) and a positive control group (cetylpyridinium chloride, final concentration of 0.1wt%) were set up, and the action time was 2min.

[0088] The test results show (as shown in Table 4) that the anti-SPA monoclonal antibody of the present invention not only has a good inhibitory effect on Staphylococcus aureus, but surprisingly, the anti-SPA monoclonal antibody of the present invention also has a strong inhibitory effect on other bacteria, which just explains the phenomenon that the monoclonal antibody has a good effect after adding it in Example 3. The reasons may be analyzed as follows:

[0089] (1) Cross-reactivity: Protein A is a highly conserved protein whose structure and function may be similar to other bacterial surface proteins. When a monoclonal antibody recognizes a specific antigen, it may accidentally bind to similar antigens on the surface of other bacteria, resulting in cross-reactions.

[0090] (2) Activation of the immune system: Antibodies can activate the host’s immune system, including the complement system, macrophages, and other immune cells, by binding to antigens on the surface of bacteria. This immune response may enhance the ability to clear streptococci and E. coli.

[0091] (3) Inhibition of bacterial adhesion: Protein A is usually involved in the adhesion process between bacteria and host cells. Antibodies can prevent bacterial adhesion and colonization by binding to these proteins, thereby affecting the infection ability of other bacteria.

[0092] (4) Impact on bacterial physiology: The binding of antibodies may interfere with the physiological functions of bacteria, such as changing the growth rate of bacteria or causing changes in cell wall synthesis, thereby inhibiting bacterial reproduction.

[0093] Table 4 Antibacterial activity test results against pathogenic bacteria

[0094]

[0095] Note: All strains are from the American type culture collection.

[0096] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An oral care solution, characterized in that: The care solution comprises the following components: Deionized water: 1000 ml, Licorice extract: 2 g, Tea tree oil: 0.5 ml, Sodium hyaluronate: 0.1 g, Citric acid: appropriate amount, Flavor: 0.5 ml food grade natural mint flavor.

2. An oral care solution, characterized in that: The care solution comprises the following components: Deionized water: 1000 ml, Licorice extract: 2 g, Tea tree oil: 0.5 ml, Sodium hyaluronate: 0.1 g, Monoclonal antibody against Staphylococcus aureus: 0.1 g, Citric acid: appropriate amount, Flavor: 0.5 ml food grade natural mint flavor.

3. The care solution according to claim 2, characterized in that: The monoclonal antibody against Staphylococcus aureus is an anti-SPA monoclonal antibody, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SPA monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

4. The care solution according to any one of claims 1 or 2, characterized in that: The citric acid is used to adjust the pH value of the care solution to 6.

5.

5. A method for preparing the care solution according to claim 2, characterized in that: The method comprises the following steps: (1) Preparation: Clean all instruments, such as stirrers, measuring cylinders, bottles, etc., and ensure they are sterile; operate in a well-ventilated environment; (2) Solvent preparation: Pour 1000 ml of deionized water into a blender and turn on the blender at medium speed to ensure uniform water flow; (3) Adding licorice extract: Slowly add 2 g of licorice extract into deionized water and stir continuously to ensure that it is completely dissolved. Generally, stir for about 5 minutes. (4) Add tea tree oil and sodium hyaluronate: Continue stirring and slowly add 0.5 ml of tea tree oil and 0.1 g of sodium hyaluronate. Keep stirring to ensure uniform dispersion. Generally, stir for about 3 to 5 minutes. (5) Addition of monoclonal antibody against Staphylococcus aureus: slowly add 0.1 g of monoclonal antibody against Staphylococcus aureus to ensure uniform mixing; (6) Adjusting pH: Gradually add citric acid, use a pH meter to monitor the pH of the solution in real time, and adjust the pH to 6.5 to ensure palatability and safety; (7) Adding flavors: Finally, add 0.5 ml of food-grade natural mint flavor and continue stirring for about 5 minutes to mix it thoroughly and ensure that the flavor is evenly distributed; (8) Filtration and storage: Aseptically filter the prepared oral care solution through a 0.22 micron sterile filter to remove any microorganisms. Bottle the filtered liquid, seal it, and store it in a cool, dry place away from light.

6. Use of the anti-SPA monoclonal antibody according to claim 3 in preparing an oral care solution.

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