Antibacterial and antioxidant toothpaste containing plant extracts and preparation method thereof

Abstract

The present invention discloses an antibacterial and antioxidant toothpaste containing plant extracts and a preparation method thereof. The toothpaste formula comprises ingredients such as green tea extract, mint extract and rosemary extract, and the toothpaste has excellent antioxidant and antibacterial properties. The preparation method of the toothpaste ensures the stability and effectiveness of plant active ingredients by adopting an appropriate extraction process, and finally forms a safe, effective and natural oral care product. Experimental results show that the toothpaste has a significant inhibitory effect on common oral pathogens and exhibits good antioxidant capacity. The toothpaste of the present invention not only improves the effect of oral health, but also meets the needs of modern consumers for natural products, and has broad market application prospects.

Description

Technical Field

[0001] The invention relates to the field of oral care, in particular to an antibacterial and antioxidant toothpaste containing plant extracts and a preparation method thereof. Background Art

[0002] Oral health is vital to overall health, and toothpaste, as an important product for daily oral care, plays an important role in preventing tooth and gum diseases. Traditional toothpastes mostly use chemically synthesized antibacterial ingredients, such as fluoride and triclosan, to inhibit the growth of bacteria in the mouth. However, long-term use of these chemical ingredients may lead to the emergence of drug-resistant strains and cause a series of side effects, such as oral mucosal irritation and allergic reactions. In addition, many consumers have higher expectations for natural ingredients in daily care products, hoping to avoid the use of synthetic chemicals and seek safer and more effective alternatives.

[0003] In recent years, plant extracts have received increasing attention due to their natural, safe and versatile properties. For example, green tea extract is rich in catechins and has significant antioxidant and antibacterial properties; peppermint extract is known for its cooling sensation and anti-inflammatory effects; and rosemary extract is believed to be effective in fighting bacteria in the mouth and promoting blood circulation. These plant ingredients not only inhibit the growth of harmful bacteria, but also provide additional antioxidant protection and slow down the cellular aging process.

[0004] Therefore, developing a toothpaste that combines these natural plant extracts can not only improve the oral care effect, but also meet consumers' demand for safe and healthy products, and has significant market potential and social value. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an antibacterial and antioxidant toothpaste containing plant extracts, which can effectively inhibit the growth of pathogenic bacteria in the oral cavity, reduce inflammation of teeth and gums, and at the same time has good antioxidant properties to protect teeth and oral health.

[0006] Therefore, the present invention discloses an antibacterial and antioxidant toothpaste containing plant extracts, the formula of the toothpaste is:

[0007] (1) Green tea extract: 10%;

[0008] (2) Peppermint extract: 10%;

[0009] (3) Rosemary extract: 10%;

[0010] (4) Calcium hydrogen phosphate: 20%;

[0011] (5) Glycerol: 15%;

[0012] (6) Aluminum silicate: 5%;

[0013] (7) Sodium lauryl sulfate: 2%;

[0014] (8) Pure water: 26.5%;

[0015] (9) Sodium benzoate: 0.5%;

[0016] (10) Mint flavor: 1%.

[0017] Preferably, the toothpaste of the present invention further comprises anti-SpaP monoclonal antibodies, and the content of the anti-SpaP monoclonal antibodies is 0.1 g / L.

[0018] Preferably, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SpaP monoclonal antibody of the present invention are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0019] Preferably, the anti-SpaP monoclonal antibody of the present invention specifically binds to the SpaP protein of Streptococcus mutans, and the position at which the anti-SpaP monoclonal antibody specifically binds to the SpaP protein is aa81-aa120, wherein the amino acid sequence of the SpaP protein at positions aa81-aa120 is shown in SEQ ID NO.2.

[0020] In another aspect, the present invention further discloses a method for preparing the toothpaste, characterized in that the method comprises the following steps:

[0021] (1) Preparation of plant extracts: 100 g each of fresh green tea leaves, mint leaves and rosemary leaves were washed and chopped; 500 ml of 60% ethanol aqueous solution were added and soaked at room temperature for 24 hours; after filtering, the extract was collected and concentrated to 250 ml to increase the extract concentration;

[0022] (2) Preparation of toothpaste: In a clean mixing container, add calcium hydrogen phosphate, slowly add 200 ml of pure water, stir evenly to ensure that the powder is completely dissolved; while stirring, gradually add concentrated plant extract, continue stirring for 5 minutes until uniform; add glycerin, aluminum silicate and sodium lauryl sulfate, continue stirring for 10 minutes to ensure that all ingredients are evenly distributed; finally, add sodium benzoate, mint flavor and anti-SpaP monoclonal antibody, and stir evenly;

[0023] (3) Filling and storage: Fill the prepared toothpaste into a sterile tube, ensure that there are no bubbles, and seal it for storage; store it in a cool and dry place, away from direct sunlight. The shelf life is 12 months.

[0024] In another aspect, the present invention also discloses the use of the anti-SpaP monoclonal antibody in the preparation of an antibacterial and antioxidant toothpaste containing plant extracts.

[0025] The toothpaste of the present invention has obvious advantages, which are mainly reflected in the following aspects:

[0026] 1. Innovative combination of natural ingredients: The present invention uses a combination of plant extracts such as green tea, mint and rosemary, which have good antibacterial and antioxidant effects in oral care. Although individual plant extracts have been used in the market, it is an innovative attempt to effectively combine these three extracts to create a composite formula with synergistic effects.

[0027] It should be noted that the researchers compared the antibacterial and antioxidant properties of adding all three ingredients at the same time and adding only one or two of them, and found that the effects were not as good as the effects of adding all three ingredients at the same time. Therefore, adding the three ingredients at the same time has a complementary effect and has obvious advantages.

[0028] 2. Highly effective antibacterial and antioxidant properties: This toothpaste is excellent in inhibiting the growth of streptococci and staphylococci, and has significant antioxidant capacity. Compared with traditional toothpaste, this invention can provide a safer and more effective oral care solution without relying on chemically synthesized antibacterial ingredients, meeting the needs of modern consumers for natural and safe products.

[0029] 3. Scientific preparation method: The preparation method of the present invention involves the rational extraction and optimization of plant extracts to ensure the biological activity of the ingredients. The plant active ingredients are extracted using a 60% ethanol aqueous solution, and the quality and effect of each batch of products are ensured through a standardized production process, which is not common in traditional toothpastes.

[0030] 4. Responding to market trends: In recent years, consumers' demand for natural products has continued to rise, especially in personal care products. The present invention combines multiple functions of antibacterial, anti-oxidation and fresh taste, which can meet the market's urgent demand for healthy and natural toothpaste and has strong market competitiveness.

[0031] 5. Comprehensive functionality: This toothpaste not only has antibacterial and antioxidant functions, but also takes into account the oral freshness effect and tooth protection, forming a multifunctional product. This comprehensive function is relatively rare in traditional toothpaste, which enhances the application value of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Anti-SpaP monoclonal antibody SDS-PAGE detection results, where 1 is the marker and 2 is the anti-SpaP monoclonal antibody. DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] Example 1: Toothpaste formulation containing plant extracts

[0036] 1. The toothpaste formula of the present invention includes the following ingredients and their proportions are as follows:

[0037] (1) Green tea extract: 10%; Green tea extract has antioxidant and antibacterial effects and can reduce the growth of bacteria in the oral cavity.

[0038] (2) Peppermint extract: 10%; Peppermint extract provides a cooling sensation, relieves oral discomfort, and also has antibacterial properties.

[0039] (3) Rosemary extract: 10%; Rosemary extract is rich in antioxidants and helps inhibit oral inflammation.

[0040] (4) Calcium hydrogen phosphate: 20%; Calcium hydrogen phosphate acts as a thickener to help form the thick texture of toothpaste, while providing calcium, which is beneficial for tooth repair.

[0041] (5) Glycerin: 15%; Glycerin acts as a humectant to help maintain the moisturizing properties of the toothpaste.

[0042] (6) Aluminum silicate: 5%; Aluminum silicate acts as an abrasive to help clean the tooth surface.

[0043] (7) Sodium lauryl sulfate: 2%; emulsifier helps the ingredients mix evenly and improves stability.

[0044] (8) Pure water: 26.5%; pure water is used to dissolve other ingredients and adjust the consistency of toothpaste.

[0045] (9) Sodium benzoate: 0.5%; preservatives prevent product deterioration and extend shelf life.

[0046] (10) Mint flavor: 1%; provides a pleasant usage experience.

[0047] Example 2: Toothpaste formulation containing plant extracts

[0048] 1. The toothpaste formula of the present invention includes the following ingredients and their proportions are as follows:

[0049] (1) Green tea extract: 10%;

[0050] (2) Peppermint extract: 10%;

[0051] (3) Rosemary extract: 10%;

[0052] (4) Calcium hydrogen phosphate: 20%;

[0053] (5) Glycerol: 15%;

[0054] (6) Aluminum silicate: 5%;

[0055] (7) Sodium lauryl sulfate: 2%;

[0056] (8) Pure water: 26.5%;

[0057] (9) Sodium benzoate: 0.5%;

[0058] (10) Mint flavor: 1%;

[0059] (11) Anti-SpaP monoclonal antibody: 0.1 g / L.

[0060] 2. Preparation and testing of anti-SpaP monoclonal antibodies

[0061] Streptococcus mutans is the main pathogen that causes tooth decay. By using monoclonal antibodies against its surface antigens, the growth or adhesion of this bacterium can be specifically inhibited to prevent the formation of caries. SpaP (also known as P1 protein) is one of the surface glycoproteins of S. mutans and is involved in the binding of bacteria to the tooth surface. Anti-SpaP monoclonal antibodies can prevent S. mutans from attaching to the tooth surface, thereby reducing the occurrence of caries, but there are currently no related monoclonal antibodies on the market. For this reason, this study specifically developed a monoclonal antibody against P1 protein.

[0062] 2.1 Preparation of anti-SpaP monoclonal antibodies

[0063] First, the amino acid sequence of the SpaP protein (as shown in SEQ ID NO.1) was analyzed, and a short peptide was designed, the position of which in the SpaP protein was aa81-aa120, and the amino acid sequence was shown in SEQ ID NO.2. The short peptide was synthesized by artificial synthesis, and then the short peptide was coupled with BSA to form a fusion protein to improve the immunogenicity.

[0064] The fusion protein was mixed evenly with an equal volume of CFA and injected intraperitoneally or subcutaneously into the mouse. The injection dose for each mouse was about 100 μg of short peptide. Two weeks later, incomplete Freund's adjuvant (IFA) was used as an adjuvant, and the fusion protein was repeated for a total of 3 immunizations, each with an interval of two weeks. After the last immunization, the spleen of the mouse was taken within 3-5 days, and sterile separation was performed. The spleen cell suspension was prepared as a single cell suspension and washed with RPMI-1640 medium. Splenocytes and myeloma cells (such as SP2 / 0) were mixed in a ratio of 10:1, and cell fusion was performed in the presence of PEG (polyethylene glycol). After fusion, the cells were cultured with RPMI-1640 medium supplemented with 20% FBS. The fused cells were inoculated in HAT selective medium and cultured to screen positive clones. About 1-2 weeks later, the culture supernatant was tested by indirect ELISA to detect whether it contained antibodies against short peptides. Select the positive wells with high antibody titers for continued culture and amplification. Finally, a hybridoma cell with the highest titer was selected, frozen and stored in liquid nitrogen.

[0065] The frozen hybridoma cell lines were expanded and cultured at a rate of 1×10 7 Hybridoma cells / mouse were intraperitoneally injected to prepare ascites, and the collected ascites was centrifuged at 4000rpm for 10min, the precipitate was discarded, and the antibody was purified after filtering with a 0.45μm / 0.22μm filter membrane. The purified monoclonal antibody (referred to as anti-SpaP monoclonal antibody) was sterilized by filtering with a 220nm filter membrane and stored at -80℃ for future use.

[0066] 2.2 Testing of anti-SpaP monoclonal antibodies

[0067] Anti-SpaP monoclonal antibody was used for SDS-PAGE detection, and the results were as follows Figure 1 As shown, the purity of SDS-PAGE can reach more than 95%.

[0068] The anti-SpaP monoclonal antibody was diluted to 1 mg / mL, and then ELISA was performed at different dilution multiples to determine the antibody titer curve. The results showed (Table 1) that the antibody could still maintain a high absorbance value at a dilution multiple of 1:10000, indicating that the monoclonal antibody had a high titer.

[0069] Table 1 ELISA titer test results

[0070] Dilution multiple 1:1000 1:5000 1:10000 1:20000 OD450nm value >4.0(The upper limit of instrument detection is 4.0) 2.67 1.89 1.23

[0071] Positive clones (1-2×10 6After collecting, centrifuge, discard the supernatant, and add 1mL Trizol to extract total RNA. According to the instructions of the kit, the total RNA is obtained by isopropanol precipitation. Using a reverse transcription kit, the extracted RNA is reverse transcribed into cDNA. Design specific primers for IgG heavy chain (VH) and light chain (VL) (as shown in Table 2). Using the above primers, the variable regions of the antibody heavy chain and light chain are amplified. The reaction conditions are pre-denaturation at 94°C for 3 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, a total of 35 cycles, and finally extended at 72°C for 10 minutes. The target band is purified using a DNA gel recovery kit to obtain purified heavy chain and light chain PCR products. The purified PCR product is sent to a sequencing company or sequenced using a Sanger sequencer. According to the sequencing results, the DNA sequence of the VH and VL regions of the antibody is obtained. The results show that the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SpaP monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0072] Table 2 Specific primers

[0073]

[0074] Example 3: Preparation of toothpaste containing plant extracts

[0075] 1. Preparation of plant extracts: Wash and chop 100 g each of fresh green tea leaves, mint leaves and rosemary leaves; add 500 ml of 60% ethanol aqueous solution and soak at room temperature for 24 hours; after filtering, collect the extract and concentrate it to 250 ml to increase the extract concentration.

[0076] 2. Preparation of toothpaste: In a clean stirring container, add calcium hydrogen phosphate, slowly add 200 ml of pure water, stir evenly, and ensure that the powder is completely dissolved; while stirring, gradually add concentrated plant extracts, continue stirring for 5 minutes until uniform; add glycerin, aluminum silicate and sodium lauryl sulfate, continue stirring for 10 minutes, and ensure that all ingredients are evenly distributed. Finally, add sodium benzoate, mint flavor and anti-SpaP monoclonal antibody (addition is required in the formula of Example 2, but not in the formula of Example 1), and stir evenly.

[0077] 3. Filling and storage: Fill the prepared toothpaste into a sterile tube, ensure there are no bubbles, and seal it for storage; store in a cool and dry place, avoid direct sunlight, the shelf life is 12 months.

[0078] Example 4: Testing of toothpaste containing plant extracts

[0079] 1. Antibacterial effect test: The antibacterial effect was tested by agar diffusion method. Common oral pathogens Streptococcus mutans and Staphylococcus aureus were selected. The toothpastes of Example 1 and Example 2 were tested together with a commercial toothpaste (Lengshenling Gum Protection Anti-Sensitive Anti-Sensitive Toothpaste) at a concentration of 10%, and compared with a control group (standard toothpaste without plant extracts). The bacterial suspension was evenly spread on the culture medium, and then a paper sheet soaked with toothpaste was placed on each culture medium and cultured at 37°C for 24 hours.

[0080] The results show (as shown in Table 3): the diameter of the antibacterial zone of the control group is 5 mm, and the antibacterial effects of the two groups of toothpastes of the present invention are better than those of the commercial ones, and the effect of the toothpaste of Example 2 is better than that of Example 1. The above results show that the toothpaste has a significant inhibitory effect on oral pathogens.

[0081] Table 3 Antibacterial effect test results

[0082] Group Diameter of Streptococcus inhibition zone (mm) Staphylococcus inhibition zone diameter (mm) Control group 5 5 Commercial toothpaste set 14 12 Example 1 Toothpaste set 20 18 Example 2 Toothpaste set 25 22

[0083] 2. Antioxidant capacity test: The antioxidant activity was evaluated by DPPH free radical scavenging method. The toothpastes of Example 1 and Example 2, together with the commercial toothpaste (Lengshenling Gum Protection Anti-Sensitive Anti-Sensitive Toothpaste), were tested at a concentration of 20 μg / ml and compared with the control group (purified water). Take an appropriate amount of sample, mix it with DPPH solution, react at room temperature for 30 minutes, and measure the absorbance change.

[0084] The calculation formula of clearance rate is: clearance rate (%) = [(control group absorbance - sample absorbance) / control group absorbance] × 100%.

[0085] The results show (as shown in Table 4) that the antioxidant capacity of the two groups of toothpastes of the present invention is better than that of the commercial ones, and the effect of the toothpaste of Example 2 is better than that of Example 1. Therefore, the toothpaste of the present invention has significant antioxidant capacity.

[0086] Table 4 Antioxidant capacity test results

[0087] Group Absorbance (A) Clearance rate (%) Control group 0.785 0 Commercial toothpaste set 0.620 21.0 Example 1 Toothpaste set 0.450 42.6 Example 2 Toothpaste set 0.320 59.2

[0088] The above results show that the toothpaste with anti-SpaP monoclonal antibody added has better effect than the toothpaste without anti-SpaP monoclonal antibody added, which indicates that the anti-SpaP monoclonal antibody prepared by the present invention has good application prospects.

[0089] 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 antibacterial and antioxidant toothpaste containing plant extracts, characterized in that: The formula of the toothpaste is: (1) Green tea extract: 10%; (2) Peppermint extract: 10%; (3) Rosemary extract: 10%; (4) Calcium hydrogen phosphate: 20%; (5) Glycerol: 15%; (6) Aluminum silicate: 5%; (7) Sodium lauryl sulfate: 2%; (8) Pure water: 26.5%; (9) Sodium benzoate: 0.5%; (10) Mint flavor: 1%; The toothpaste also includes anti-SpaP monoclonal antibodies, and the content of the anti-SpaP monoclonal antibodies is 0.1 g / L; The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-SpaP monoclonal antibody are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; The anti-SpaP monoclonal antibody specifically binds to the SpaP protein of Streptococcus mutans, and the position where the anti-SpaP monoclonal antibody specifically binds to the SpaP protein is aa81-aa120, wherein the amino acid sequence of the SpaP protein at the position aa81-aa120 is shown in SEQ ID NO.

2.

2. Use of the anti-SpaP monoclonal antibody according to claim 1 in the preparation of an antibacterial and antioxidant toothpaste containing plant extracts.

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