Brazilian green propolis gum-protecting toothpaste as well as preparation method and application thereof

Abstract

The invention discloses Brazilian green propolis gingiva-protecting toothpaste as well as a preparation method and an effect thereof. The process is simple, the components are safe, and through three-batch verification, the formula and the process can achieve stable production; the toothpaste can relieve bad breath, care periodontium, reduce gingival inflammation and maintain oral health, and is worthy of being used for a long time.

Description

Technical Field

[0001] The invention relates to the technical field of toothpaste, and in particular to a Brazilian green propolis gum-protecting toothpaste, a preparation method and application thereof. Background Art

[0002] As residents' income increases, the oral consumption concept of Chinese residents has improved, and they are paying more and more attention to oral health management, and the demand is growing. Toothpaste is a consumer product used by residents in daily life, and there are many types. As a type of toothpaste, the market for gum-protecting toothpaste will increase as consumers pay more attention to oral health and the demand for functional toothpaste is diversified. There are many types of gum-protecting toothpaste on the market, but there are very few toothpastes made from Brazilian green propolis as a raw material. In view of the residents' attention to oral health and the development of diversified gum-protecting toothpaste, the present invention provides a method for preparing Brazilian green propolis gum-protecting toothpaste, a process and its application. Summary of the Invention

[0003] The purpose of the present invention is to provide a toothpaste formula and process with simple process and easy method to solve the defects and shortcomings of the existing technology.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] A Brazilian green propolis gum protection toothpaste, the innovation of which lies in that it is composed of the following raw materials in percentage by weight: 60%-70% sorbitol, 10%-20% hydrated silica, 1%-5% polyethylene glycol-8, 0.1%-1.5% ethanol, 0.5%-3% sodium lauryl sulfate, 0.1%-1% cellulose gum, 0.1%-1% Chondrus crispus extract, 0.1%-1% chlorhexidine digluconate, 0.1%-0.5% trichlorogalactose, 0.1%-0.5% propolis extract, 0.01%-0.2% tetrasodium pyrophosphate, 0.01%-0.1% o-cymene-5-ol, black tea (CAMELLIA The invention is composed of 0.01%-0.1% of SINENSIS extract, 0.01%-0.1% of dipotassium glycyrrhizate, 0.01%-0.1% of chlorophyllin-copper complex, 0.01%-0.1% of tocopherol, edible essence and purified water.

[0006] A preparation method of Brazilian green propolis gum-protecting toothpaste, the innovation of which lies in the following steps:

[0007] S1. According to the formula ratio, heat purified water to about 35°C, add 0.1%-0.5% trichlorogalactose, 0.01%-0.2% tetrasodium pyrophosphate, 0.01%-0.1% dipotassium glycyrrhizate, 0.01%-0.1% chlorophyllin-copper complex, and 0.01%-0.1% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I;

[0008] S2. Mixing 0.1%-1% Chondrus Crispus extract, 0.1%-1% cellulose gum, 10%-20% hydrated silica, and 0.5%-3% sodium lauryl sulfate to obtain a mixture II.

[0009] S3, dissolving 0.1%-0.5% propolis extract in 0.1%-1.5% ethanol to obtain mixture III;

[0010] S4. Dissolve 0.01%-0.1% o-cymene-5-ol and 0.01%-0.1% tocopherol in 1%-5% polyethylene glycol-8% to obtain mixture IV;

[0011] S5. According to the formula ratio, add mixture I to the vacuum pot, then add 60%-70% sorbitol, and stir for 5 minutes; then add mixture III, mixture IV and 0.1%-1% chlorhexidine digluconate into the vacuum pot and stir for 10 minutes; then add mixture II, stir and homogenize for 30 minutes, and finally add flavor, vacuum, stir and homogenize for 30 minutes to form a paste.

[0012] The beneficial effects of the present invention are:

[0013] The invention provides an oral care product with a simple process and easy implementation. Experiments have shown that the product has the effect of reducing gingivitis. The invention not only improves the basic function of toothpaste, which is only to clean teeth, but also reduces bad breath, protects periodontal tissues, and reduces gingivitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the ELISA standard curve of TNF-α content of the present invention;

[0015] Figure 2 It is the TNF-α content of each group of the present invention. DETAILED DESCRIPTION

[0016] The following examples further illustrate the present invention.

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0018] Example 1

[0019] A Brazilian green propolis gum protection toothpaste is composed of the following raw materials in percentage by weight: 55%-65% sorbitol, 10%-20% hydrated silica, 1%-3% polyethylene glycol-8, 0.1%-1.5% ethanol, 0.5%-3% sodium lauryl sulfate, 0.1%-1% cellulose gum, 0.1%-1% Chondrus crispus extract, 0.1%-1% chlorhexidine digluconate, 0.1%-0.5% trichlorogalactose, 0.1%-0.5% propolis extract, 0.01%-0.2% tetrasodium pyrophosphate, 0.01%-0.1% o-cymene-5-ol, black tea (CAMELLIA The invention is composed of 0.01%-0.1% of SINENSIS extract, 0.01%-0.1% of dipotassium glycyrrhizate, 0.01%-0.1% of chlorophyllin-copper complex, 0.01%-0.1% of tocopherol, edible essence and purified water.

[0020] In this embodiment:

[0021] 1. Sorbitol: humectant, solubility aid, pharmaceutical excipient.

[0022] 1.1 Sorbitol is an excellent humectant that absorbs moisture from the air and locks in moisture, preventing toothpaste from drying out and clumping. This keeps the toothpaste moist during use, enhancing the user experience. Sorbitol also lowers the freezing point of toothpaste, preventing it from hardening or freezing in low temperatures, thereby improving its stability.

[0023] 1.2 Sorbitol has a slightly sweet taste that enhances the taste of toothpaste, making it smoother and more pleasant without causing tooth decay. This sweetness makes it one of the most common sweeteners in toothpaste.

[0024] 2. Hydrated silica: abrasive, thickener

[0025] 2.1 Hydrated silica acts as an abrasive and thickener in toothpaste. Its fine particles come into contact with the tooth surface, effectively removing tartar, stains, and plaque, thereby enhancing cleaning effectiveness. This action is gentle and does not cause excessive abrasion to teeth, making it suitable for daily use.

[0026] 3. Polyethylene glycol-8: lubricant, solubilizer

[0027] 3.1 Polyethylene glycol-8 is a common lubricant that can reduce friction between toothpaste and the teeth and mouth, thereby improving the user experience. For example, it can effectively reduce the stickiness of toothpaste, making it easier to apply and rinse, while also protecting the oral mucosa from irritation.

[0028] 3.2 Polyethylene glycol-8 has good solvent properties and can dissolve other ingredients, such as fluoride, so that they can play a better role in the mouth. For example, it can help the fluoride in fluoride toothpaste better penetrate the tooth surface and enhance the tooth's anti-caries ability.

[0029] 4. Ethanol: solvent, preservative

[0030] 4.1 Ethanol is a common preservative with significant antibacterial and antimicrobial effects. For example, ethanol extracts in toothpaste have a strong inhibitory effect on oral bacteria (such as Staphylococcus aureus and Streptococcus), effectively reducing the bacterial population in the mouth, thereby improving oral hygiene and preventing diseases such as dental plaque and gingivitis. Furthermore, ethanol is also used in certain special-purpose toothpastes, such as whitening toothpaste, to reduce oral odor by inhibiting bacterial growth.

[0031] 5. Sodium lauryl sulfate: foaming agent

[0032] 5.1 Sodium lauryl sulfate can effectively remove dirt and food residues on the surface of teeth, soften tartar and maintain oral hygiene.

[0033] 5.2 Foaming ability: It can significantly increase the production of foam, making the brushing process more comfortable and helping to remove plaque and tartar.

[0034] 5.3 Emulsification: Sodium lauryl sulfate can mix oil and water, thereby better cleaning teeth and mouth.

[0035] 6. Cellulose gum: thickener

[0036] 6.1 Cellulose gum is a commonly used thickener in toothpaste, which can significantly increase the viscosity and fluidity of toothpaste. This makes the toothpaste easier to apply and use, and also helps it adhere better to the toothbrush, preventing it from running off during use.

[0037] 7. Chondrus Crispus Extract: Thickener

[0038] 7.1 Chondrus crispus extract not only enhances toothpaste performance (e.g., thickening and moisturizing) but also exhibits multiple benefits, including anti-inflammatory, antibacterial, antioxidant, cleansing, whitening, and gum repair. These properties make it a versatile and safe toothpaste ingredient suitable for widespread use in oral care products.

[0039] 8. Chlorhexidine digluconate: preservative

[0040] 8.1 The main function of chlorhexidine digluconate in toothpaste is to reduce the number of bacteria in the oral cavity through its antibacterial properties, prevent the occurrence of caries and periodontal disease, and act as a preservative to extend the shelf life of the product.

[0041] 9. Trichlorogalactose: flavoring agent

[0042] 9.1 Trichlorogalactose is a high-intensity sweetener, approximately 600 times sweeter than sucrose, and is calorie-free. It provides a pure sweetness without any off-flavor or bitterness, making it widely used as a sweetener in toothpaste. It significantly improves the taste of toothpaste, making it more comfortable and pleasant, thereby enhancing the user experience.

[0043] 10. Propolis extract: antibacterial, anti-inflammatory, antioxidant

[0044] 10.1 Propolis extract has significant antibacterial activity, inhibiting the growth of a variety of oral pathogens. Studies have shown that propolis extract exhibits significant antibacterial activity against a variety of bacteria, including Streptococcus mutans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, with its antibacterial activity comparable to that of triclosan. Furthermore, propolis extract inhibits the acid production of cariogenic bacteria, such as Streptococcus mutans, thereby reducing the incidence of dental caries.

[0045] 10.2 Propolis extract is rich in flavonoids and phenolic acids, which have anti-inflammatory properties and can alleviate the symptoms of gingivitis and periodontitis. For example, propolis extract can reduce the release of inflammatory mediators such as prostaglandins and leukotrienes, thereby accelerating wound healing.

[0046] 10.3 Propolis extract is rich in antioxidant ingredients, such as flavonoids and polyphenols, which can scavenge free radicals and protect oral tissues from oxidative damage.

[0047] 11. Sodium pyrophosphate: pH buffer, pharmaceutical excipient.

[0048] 11.1 Quality improvers have the function of increasing the complex metal ions, pH value, and ionic strength of food, thereby improving the binding force and water holding capacity of food.

[0049] 12. O-cymene-5-ol: preservative

[0050] 12.1-O-cymene-5-ol is a broad-spectrum antibacterial agent that effectively inhibits a variety of oral bacteria, including Staphylococcus aureus, Escherichia coli, and Streptococcus mutans. It can penetrate deep into dental plaque and inhibit bacterial growth, thereby reducing plaque formation and preventing oral diseases such as gingivitis and periodontal disease.

[0051] 13. Camellia sinensis extract: antibacterial, anti-inflammatory

[0052] 13.1 Black tea extract in toothpaste not only provides multiple benefits such as antibacterial, anti-inflammatory, anti-caries, whitening, and breath-improving properties, but also enhances the product's taste and user experience. It is a natural ingredient that combines health benefits with practical benefits. These properties have led to its widespread use in modern toothpaste products.

[0053] 14. Dipotassium glycyrrhizate: antibacterial, anti-inflammatory

[0054] 14.1 The primary effects of dipotassium glycyrrhizate in toothpaste include antibacterial, anti-inflammatory, ulcer healing, detoxification, and anti-allergic properties. These properties make it an important ingredient for improving oral health and, due to its high safety, it is widely used in toothpaste formulations.

[0055] 15. Chlorophyllin-copper complex: colorant

[0056] 15.1 Chlorophyllin-copper complex is a natural green pigment that can be extracted from plants. It is used as a colorant in toothpaste to give it a green color, thereby enhancing the product's appearance.

[0057] 16. Tocopherol: Antioxidant

[0058] 16.1 Tocopherol is primarily used in toothpaste as an antioxidant and soothing ingredient, focusing on maintaining oral soft tissue health rather than directly affecting tooth cleaning or whitening. Choosing a toothpaste containing this ingredient may be more beneficial for people who are prone to gingivitis or oral sensitivity.

[0059] 17. Flavor: Flavoring agent used to cover up the bad smell of raw materials.

[0060] 18. Purified water uses secondary purified water with low ion content.

[0061] The invention adopts hydrated silica as an abrasive, cellulose gum and Chondrus crispus extract as thickeners, sorbitol and polyethylene glycol as moisturizers, ethanol, chlorhexidine and cymene as preservatives, and black tea extract, dipotassium glycyrrhizate and propolis extract as effective ingredients to make a paste.

[0062] Example 2

[0063] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0064] S1. Heat purified water to about 35°C according to the formula ratio, add trichlorogalactose, tetrasodium pyrophosphate, dipotassium glycyrrhizate, chlorophyllin-copper complex, and black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain mixture I.

[0065] S2. Evenly stir the Chondrus Crispus extract, cellulose gum, hydrated silica, and sodium lauryl sulfate to obtain a mixture II.

[0066] S3. Dissolve the propolis extract in ethanol to obtain mixture III.

[0067] S4. Dissolve o-cymene-5-ol and tocopherol with polyethylene glycol-8 to obtain mixture IV.

[0068] S5. Add Mixture I to the vacuum pot according to the formula ratio, then add sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV and chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, stir and homogenize for 30 minutes to form a paste.

[0069] Example 3

[0070] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0071] S1. According to the formula ratio, heat purified water to about 35°C, add 0.1% trichlorogalactose, 0.01% tetrasodium pyrophosphate, 0.01% dipotassium glycyrrhizate, 0.01% chlorophyllin-copper complex, and 0.01% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I.

[0072] S2. Mix 0.1% Chondrus Crispus extract, 0.1% cellulose gum, 10% hydrated silica, and 0.5% sodium lauryl sulfate to obtain a mixture II.

[0073] S3. Dissolve 0.1% propolis extract in 0.1% ethanol to obtain mixture III.

[0074] S4. Dissolve 0.01% o-cymene-5-ol and 0.01% tocopherol in 1% polyethylene glycol-8 to obtain mixture IV.

[0075] S5. Add Mixture I to the vacuum pot according to the formula ratio, then add 55% sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV, and 0.1% chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, and stir and homogenize for 30 minutes to form a paste.

[0076] Example 4

[0077] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0078] S1. According to the formula ratio, heat purified water to about 35°C, add 0.5% trichlorogalactose, 0.2% tetrasodium pyrophosphate, 0.1% dipotassium glycyrrhizate, 0.1% chlorophyllin-copper complex, and 0.1% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I.

[0079] S2. Mix 1% Chondrus Crispus extract, 1% cellulose gum, 20% hydrated silica, and 3% sodium lauryl sulfate to obtain a mixture II.

[0080] S3. Dissolve 0.5% propolis extract in 1.5% ethanol to obtain mixture III.

[0081] S4. Dissolve 0.1% o-cymene-5-ol and 0.1% tocopherol in 3% polyethylene glycol-8 to obtain mixture IV.

[0082] S5. Add Mixture I to the vacuum pot according to the recipe ratio, then add 65% sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV, and 1% chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, and stir and homogenize for 30 minutes to form a paste.

[0083] Example 5

[0084] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0085] S1. According to the formula ratio, heat purified water to about 35°C, add 0.3% trichlorogalactose, 0.1% tetrasodium pyrophosphate, 0.05% dipotassium glycyrrhizate, 0.03% chlorophyllin-copper complex, and 0.05% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I.

[0086] S2. 0.5% Chondrus Crispus extract, 0.5% cellulose gum, 15% hydrated silica, and 1.6% sodium lauryl sulfate were uniformly stirred to obtain a mixture II.

[0087] S3. Dissolve 0.2% propolis extract in 0.75% ethanol to obtain mixture III.

[0088] S4. Dissolve 0.05% o-cymene-5-ol and 0.03% tocopherol in 3% polyethylene glycol-8 to obtain mixture IV.

[0089] S5. Add Mixture I to the vacuum pot according to the formula ratio, then add 58% sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV, and 0.5% chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, and stir and homogenize for 30 minutes to form a paste.

[0090] Example 6

[0091] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0092] S1. Heat purified water to about 35°C according to the formula ratio, add 0.3% trichlorogalactose, 0.1% tetrasodium pyrophosphate, 0.05% dipotassium glycyrrhizate, 0.06% chlorophyllin-copper complex, and 0.05% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I.

[0093] S2. 0.3% Chondrus Crispus extract, 0.6% cellulose gum, 18% hydrated silica, and 1% sodium lauryl sulfate were uniformly stirred to obtain a mixture II.

[0094] S3. Dissolve 0.2% propolis extract in 1% ethanol to obtain mixture III.

[0095] S4. Dissolve 0.05% o-cymene-5-ol and 0.03% tocopherol in 3% polyethylene glycol-8 to obtain mixture IV.

[0096] S5. Add Mixture I to the vacuum pot according to the recipe ratio, then add 62% sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV, and 0.25% chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, and stir and homogenize for 30 minutes to form a paste.

[0097] Example 7

[0098] A preparation method of Brazilian green propolis gum protection toothpaste comprises the following steps:

[0099] S1. According to the formula ratio, heat purified water to about 35°C, add 0.15% trichlorogalactose, 0.1% tetrasodium pyrophosphate, 0.05% dipotassium glycyrrhizate, 0.04% chlorophyllin-copper complex, and 0.05% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I.

[0100] S2. 0.3% Chondrus Crispus extract, 0.3% cellulose gum, 18% hydrated silica, and 1% sodium lauryl sulfate were uniformly stirred to obtain a mixture II.

[0101] S3. Dissolve 0.2% propolis extract in 1.2% ethanol to obtain mixture III.

[0102] S4. Dissolve 0.1% o-cymene-5-ol and 0.1% tocopherol in 3% polyethylene glycol-8 to obtain mixture IV.

[0103] S5. Add Mixture I to the vacuum pot according to the formula ratio, then add 64% sorbitol and stir for 5 minutes. Then add Mixture III, Mixture IV, and 0.25% chlorhexidine digluconate to the vacuum pot and stir for 10 minutes. Then add Mixture II and stir and homogenize for 30 minutes. Finally, add the flavoring, vacuumize, and stir and homogenize for 30 minutes to form a paste.

[0104] Example 8

[0105] Three batches of a Brazilian green propolis gum protection toothpaste were produced, including the following:

[0106] 1. The production formula of three batches is shown in Table 1:

[0107]

[0108]

[0109] 2. The production information of the three batches is shown in Table 2:

[0110]

[0111] 3. The production process is as shown in Example 2

[0112] 4. Inspection data of three batches of semi-finished products are shown in Table 3:

[0113]

[0114]

[0115] 5. Inspection data of three batches of finished products are shown in Table 4:

[0116]

[0117] 6. Conclusion

[0118] The production of three consecutive batches can prove the stability of the process and the rationality of the formula.

[0119] Example 9

[0120] The efficacy of reducing gum inflammation, the test results are as follows:

[0121] 1. Test materials

[0122] 1.1 Instruments and Materials Cells: Human gingival fibroblasts, purchased from Guangdong Boxi Biotechnology Co., Ltd. Main reagents: Gingival fibroblast culture system TNF-α inflammatory factor ELISA kit

[0123] Main instruments: CO2 incubator, clean bench, inverted microscope, microplate reader

[0124] Instrument name Instrument model Instrument Manufacturer <![CDATA[CO2 Incubator]]> MW160 Guansen Biotechnology (Shanghai) Co., Ltd. Clean bench SW-CJ-1FD Suzhou Su Jie Inverted microscope TS100 NIKON microplate reader SpectraMAX i3 Molecular Devices

[0125] 1.2 Positive Control

[0126] Name: Dexamethasone

[0127] Batch number: C14206294

[0128] Manufacturer: MACKLIN

[0129] Solvent: DMSO

[0130] Concentration: 100 μg / mL

[0131] 1.3 Negative control (stimulant)

[0132] Name: LPS lipopolysaccharide

[0133] Batch number: #0000108436

[0134] Manufacturer: Sigma

[0135] Solvent: PBS

[0136] Concentration: 1 μg / mL

[0137] 1.4 Preparation of positive and negative groups

[0138] Positive control group: 10 mg of dexamethasone was dissolved in 1 mL of DMSO solution to form the dexamethasone stock solution with a concentration of 10 mg / mL; the dexamethasone stock solution was aspirated and diluted 1:100 to 100 μg / mL using the gingival fibroblast culture system containing LPS as the positive control working solution.

[0139] Negative control group (stimulant): 10 mg LPS was dissolved in 2 mL DMSO solution to form LPS stock solution with a concentration of 5 mg / mL; the LPS stock solution was aspirated and diluted to 1 μg / mL at 1:5000 using the gingival fibroblast culture system.

[0140] 2. Test methods

[0141] 2.1 Cytotoxicity test

[0142] 2.1.1 Cell seeding: Cells were seeded into 96-well plates at a seeding density of 1×104 cells / well and incubated overnight in an incubator (37°C, 5% CO2).

[0143] 2.1.2 Experimental grouping: The experiment set up zero adjustment group, solvent control group, positive control group, and sample group. In the sample group, the samples were set at 8 concentration gradients.

[0144] Three replicate wells were set up for each concentration gradient.

[0145] 2.1.3 Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (1).

[0146] Table 1 Test concentration setting table

[0147]

[0148] 2.1.4 Sampling: Sampling was performed when the cell plating rate in the 96-well plate reached 40%-60%. The zero adjustment group was seeded with 200 μL of gingival fibroblast culture system per well; the solvent control group was seeded with 200 μL of gingival fibroblast culture system per well; the positive control group was seeded with 200 μL of gingival fibroblast culture system containing 10% DMSO per well; and the sample group was seeded with 200 μL of gingival fibroblast culture system containing the corresponding concentration of sample per well. After sampling, the 96-well plate was placed in an incubator (37°C, 5% CO2) for incubation.

[0149] 2.1.5 Detection: After incubating the cells for 24 h, discard the supernatant, add MTT working solution (0.5 mg / mL), incubate at 37°C in the dark for 4 h, discard the supernatant, add 150 μL DMSO to each well, and read the OD value at 490 nm.

[0150] 2.1.6 Calculation of relative cell viability:

[0151]

[0152] 2.2 Efficacy test

[0153] 2.2.1 Trial Grouping

[0154] The specific settings of the experimental groups are shown in Table 2.

[0155] Table 2 Experimental groups

[0156]

[0157] 2.2.2 Cell inoculation: Cells in logarithmic growth phase were collected and seeded into 96-well plates at a cell density of 1×104 cells / well. The plates were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0158] 2.2.3 Sampling: Sampling was performed when the cell plating rate in the 96-well plate reached 40%-60%. The blank control group consisted of a gingival fibroblast culture system (200 μL per well), the negative control group consisted of a gingival fibroblast culture system containing LPS (200 μL per well), the positive control group consisted of a gingival fibroblast culture system containing a positive control and LPS (200 μL per well), and the sample group consisted of a gingival fibroblast culture system containing a certain concentration of the test substance and LPS (200 μL per well). After sampling, the 96-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0159] 2.2.4 ELISA Assay: After incubation, collect the cell culture medium into a 1.5 mL Eppendorf tube and centrifuge at 1000 × g at 4°C. The supernatant is used for TNF-α detection. For subsequent experimental procedures, refer to the corresponding kit instructions.

[0160] 3. Test results and analysis

[0161] 3.1 Cytotoxicity results

[0162] Table 3 Cytotoxicity results

[0163]

[0164] 3.2 Standard curve

[0165] Draw a standard curve with the concentration of the standard as the ordinate and the OD value as the abscissa. Based on the OD450 value of the sample, find the corresponding concentration and calculate the TNF-α content of the sample. The average of the three replicate wells in each group is used as the final TNF-α content result.

[0166] 3.3 Results and their judgment

[0167] Inhibition rate (%) = (1-T / C) × 100%

[0168] T—average value of TNF-α content of the test substance;

[0169] C—Average TNF-α content in negative control.

[0170] Table 4 TNF-α detection results

[0171] Group TNF-α content mean ± standard deviation (SD) Inhibition rate (%) P-value Negative control 515.18±11.37 —— —— 1000 μg / mL 317.08±14.55 38.45 ** 500 μg / mL 358.30±4.52 30.45 ** 250 μg / mL 367.51±14.57 28.66 ** Positive control 348.39±7.79 32.38 **

[0172] Note: Compared with the negative control group, there is a significant difference, *P < 0.05, **P < 0.01. No significant difference, P > 0.05, indicated by "ns".

[0173] 4. Conclusion

[0174] When the test sample concentration was between 250 μg / mL and 1000 μg / mL, the TNF-α content in each group decreased compared with the negative control, and there was a significant difference (P<0.05), which showed that the test sample had the effect of inhibiting TNF-α and reducing gingival inflammation.

[0175] The above description is only a preferred embodiment of the present invention, which is only used to illustrate the technical solution of the present invention and is not intended to limit the present invention. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A Brazilian green propolis gum protection toothpaste, characterized in that: The invention is composed of the following raw materials in percentage by weight: 55%-65% sorbitol, 10%-20% hydrated silica, 81%-3% polyethylene glycol, 0.1%-1.5% ethanol, 0.5%-3% sodium lauryl sulfate, 0.1%-1% cellulose gum, 0.1%-1% CHONDRUS CRISPUS extract, 0.1%-1% chlorhexidine digluconate, 0.1%-0.5% trichlorogalactose, 0.1%-0.5% propolis extract, 0.01%-0.2% tetrasodium pyrophosphate, 0.01%-0.1% o-cymene-5-ol, black tea (CAMELLIA The invention is composed of 0.01%-0.1% of SINENSIS extract, 0.01%-0.1% of dipotassium glycyrrhizate, 0.01%-0.1% of chlorophyllin-copper complex, 0.01%-0.1% of tocopherol, edible essence and purified water.

2. A method for preparing the Brazilian green propolis gum protection toothpaste according to claim 1, characterized in that: The following steps are involved: S1. According to the formula ratio, heat purified water to about 35°C, add 0.1%-0.5% trichlorogalactose, 0.01%-0.2% tetrasodium pyrophosphate, 0.01%-0.1% dipotassium glycyrrhizate, 0.01%-0.1% chlorophyllin-copper complex, and 0.01%-0.1% black tea (CAMELLIA SINENSIS) extract, and stir for 30-60 minutes to obtain Mixture I; S2. Mixing 0.1%-1% Chondrus Crispus extract, 0.1%-1% cellulose gum, 10%-20% hydrated silica, and 0.5%-3% sodium lauryl sulfate to obtain a mixture II. S3, dissolving 0.1%-0.5% propolis extract in 0.1%-1.5% ethanol to obtain mixture III; S4. Dissolve 0.01%-0.1% o-cymene-5-ol and 0.01%-0.1% tocopherol in 1%-5% polyethylene glycol-8 to obtain mixture IV; S5. According to the formula ratio, add mixture I to the vacuum pot, then add 55%-65% sorbitol, and stir for 5 minutes; then add mixture III, mixture IV and 0.1%-1% chlorhexidine digluconate into the vacuum pot and stir for 10 minutes; then add mixture II, stir and homogenize for 30 minutes, and finally add flavor, vacuum, stir and homogenize for 30 minutes to form a paste.

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