Composition with bacteriostatic, anti-inflammatory and / or oral cavity repairing effects and application of composition in toothpaste

Abstract

The invention relates to a composition with bacteriostatic, anti-inflammatory and / or oral cavity repairing effects and application of the composition in toothpaste, and belongs to the technical field of daily chemicals. The composition disclosed by the invention is prepared from the following components in parts by weight: 0.04 to 0.06 part of pyrroloquinoline quinone and 0.2 to 0.3 part of carboxymethyl chitosan. Based on the anti-oxidative stress regulation and control capability of pyrroloquinoline quinone and the broad-spectrum antibacterial synergistic effect of carboxymethyl chitosan, pyrroloquinoline quinone and carboxymethyl chitosan are compounded and applied to a toothpaste formula system, so that the toothpaste has the effects of inhibiting bacteria, resisting inflammation, proliferating and repairing gingival fibroblasts and regulating the composition and diversity of oral microorganisms. According to the composition disclosed by the invention, through a multi-mechanism synergistic effect, an oral health maintenance system integrating bacteriostatic protection, inflammation regulation and control and mucous membrane repair is constructed, and the compound requirements of modern consumers on oral microenvironment regulation and control, mucous membrane barrier strengthening and long-term health management are accurately met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of daily chemicals, in particular to a composition with bacteriostatic, anti-inflammatory and / or oral repair effects and its application in toothpaste. BACKGROUND

[0002] Pyrroloquinoline quinone (PQQ) is an aromatic tricyclic o-quinone compound with a molecular formula of C 14 H6N2O8 and a molecular weight of 330.206. It is the third coenzyme discovered after flavin nucleotides and nicotinamide nucleotides. PQQ is a water-soluble B vitamin that exists in almost all foods. Mammals cannot synthesize PQQ themselves, but PQQ is widely found in nature, such as soybeans, tea, green peppers, coriander, kiwis, etc. Therefore, PQQ also exists in trace amounts in humans and other animals, and humans mainly rely on food to obtain PQQ. PQQ has anti-inflammatory, antioxidant, anti-aging, mitochondrial growth promotion, immune enhancement, liver damage prevention, energy metabolism regulation, osteoporosis repair, and cell damage reduction effects. Even some studies have found that PQQ also has certain anti-cancer functions, and has broad application prospects in the fields of medicine, health products, food, and cosmetics, etc. It is a high-value raw material.

[0003] Carboxymethyl chitosan (CMCS) is a derivative of chitosan, which is derived from natural polysaccharide extracts from the shells of crustaceans such as shrimp and crabs. After alkaline treatment to remove acetyl groups, chitosan is converted to chitosan. Subsequently, chitosan reacts with chloroacetic acid in an alkaline environment to introduce carboxymethyl groups, thereby generating carboxymethyl chitosan. By introducing carboxymethyl groups through chemical modification, carboxymethyl chitosan exhibits superior solubility and biocompatibility, and has biological activities such as antibacterial, antioxidant, and wound healing promotion. Carboxymethyl chitosan (CMCS) has excellent biocompatibility, antibacterial properties, antioxidant properties, and tissue repair capacity, and is widely used in the fields of medicine, cosmetics, and food, etc.

[0004] Traditional toothpaste may disrupt the balance of oral microorganisms, leading to a decrease in beneficial bacteria and increasing the risk of oral problems. Traditional toothpaste mainly focuses on cleaning and prevention, and lacks components that promote oral tissue repair. Traditional toothpaste relies heavily on chemically synthesized ingredients and lacks natural extracts, which may not be attractive to consumers seeking natural products. The use of some Chinese herbal medicines in toothpaste may also result in undesirable consequences such as toothpaste discoloration, taste change, and poor stability. There is currently no related research on the simultaneous application of pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in toothpaste products. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide a composition with bacteriostatic, anti-inflammatory and / or oral repair effects and its application in toothpaste.

[0006] The present application applies pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in toothpaste formulations, which can give the toothpaste a proliferation repair effect on gingival fibroblasts (HGF). Gingival fibroblasts (HGF) are located in the gingival tissue and mainly distributed in the connective tissue of the gums. Gingival fibroblasts (HGF) play a key role in gum health and disease, especially in diseases such as periodontitis, and their abnormal function can lead to tissue destruction.

[0007] The application of pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in toothpaste not only maintains gum health but also effectively maintains oral microecological balance. Oral microecological balance can prevent dental caries, periodontal disease and oral infection, support immune function, promote digestive function, and have a positive impact on overall health.

[0008] And the pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) complement each other and have a synergistic effect, and the toothpaste passes the stability test of aging at 45℃ for 3 months, without discoloration and odor, with good stability.

[0009] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0010] In a first aspect, the present application provides a composition with bacteriostatic, anti-inflammatory and / or oral repair effects, which comprises 0.04-0.06 parts by weight of pyrroloquinoline quinone (PQQ) and 0.2-0.3 parts by weight of carboxymethyl chitosan (CMCS).

[0011] The present application combines pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) as active substances of toothpaste, and the combination of the two has a synergistic effect on bacteriostatic effect.

[0012] The present application is based on the synergistic effect of the antioxidant stress regulation ability of pyrroloquinoline quinone (PQQ) and the broad-spectrum antibacterial effect of CMCS. The two are compounded in toothpaste formula system. The targeted inhibition ability of the toothpaste on cariogenic bacteria such as Streptococcus mutans, Porphyromonas gingivalis and the like is measured by antibacterial experiment. The anti-inflammatory effect experiment shows that the toothpaste sample containing pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) has a certain anti-inflammatory effect. The toothpaste is tested by oral repair experiment, and it is found that the toothpaste has proliferation and repair effect on gingival fibroblasts. The toothpaste is tested by micro-ecological evaluation, and the product has the effect of affecting the composition and diversity of oral microorganisms. The innovative formula realizes the synergistic effect of multiple mechanisms, and builds an oral health maintenance system integrating antibacterial protection, inflammation regulation and mucosal repair, which accurately matches the complex needs of modern consumers for oral microenvironment regulation, mucosal barrier strengthening and long-term health management.

[0013] As a preferred embodiment of the composition of the present application, the composition comprises pyrroloquinoline quinone (PQQ) 0.05-0.06 parts and carboxymethyl chitosan (CMCS) 0.25-0.3 parts by weight. Under this range, the antibacterial and anti-inflammatory effects are better.

[0014] As a preferred embodiment of the composition of the present application, the composition comprises pyrroloquinoline quinone (PQQ) 0.05 parts and carboxymethyl chitosan (CMCS) 0.25 parts by weight. Under this proportion, the antibacterial and anti-inflammatory effects are better.

[0015] As a preferred embodiment of the composition of the present application, the composition comprises pyrroloquinoline quinone (PQQ) 0.04-0.05 parts and carboxymethyl chitosan (CMCS) 0.2-0.25 parts by weight. Under this range, the oral repair effect is better.

[0016] As a preferred embodiment of the composition of the present application, the composition comprises pyrroloquinoline quinone (PQQ) 0.04 parts and carboxymethyl chitosan (CMCS) 0.2 parts by weight. Under this proportion, the oral repair effect is better.

[0017] As a preferred embodiment of the composition of the present application, the molecular weight of the carboxymethyl chitosan (CMCS) is 5000-10000 Da, and the molecular size is moderate, which can effectively penetrate the bacterial cell wall and penetrate into the intracellular, thereby significantly improving the antibacterial efficiency. The degree of substitution of carboxymethyl of CMCS is 0.8-1.2, which optimizes the charge distribution and hydrophilicity of CMCS, not only enhances the solubility in aqueous system, but also further activates the antibacterial activity of targeted destruction of bacterial membrane structure.

[0018] In the second aspect, the present application provides the use of the above-mentioned composition in the preparation of toothpaste.

[0019] In a third aspect, the present application provides a toothpaste comprising 0.24-0.36% of the above-mentioned composition and 99.64-99.76% of toothpaste auxiliaries, by mass percentage.

[0020] Pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) are two substances with unique biological activity, which are added in the toothpaste formula, and can achieve more efficient oral health maintenance, and meet the needs of consumers for multifunctional oral care products. The toothpaste is tested by bacteriostatic experiment, and it is proved that the toothpaste has good inhibitory effect on Streptococcus mutans and Porphyromonas gingivalis; the anti-inflammatory effect experiment proves that the toothpaste sample containing pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) has certain anti-inflammatory effect; the toothpaste is tested by oral repair experiment, and it is proved that the toothpaste has proliferation and repair effect on gingival fibroblasts. The toothpaste is tested by micro-ecological evaluation, and the product has the effect of affecting the composition and diversity of oral microorganisms, and can adjust the composition and diversity of oral microorganisms.

[0021] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.04-0.06% of pyrroloquinoline quinone (PQQ), 0.2-0.3% of carboxymethyl chitosan (CMCS) and 99.64-99.76% of toothpaste auxiliaries, by mass percentage.

[0022] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.05-0.06% of pyrroloquinoline quinone (PQQ), 0.25-0.3% of carboxymethyl chitosan (CMCS) and 99.64-99.7% of toothpaste auxiliaries, by mass percentage.

[0023] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.05% of pyrroloquinoline quinone (PQQ), 0.25% of carboxymethyl chitosan (CMCS) and 99.7% of toothpaste auxiliaries, by mass percentage.

[0024] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.04% of pyrroloquinoline quinone (PQQ), 0.2% of carboxymethyl chitosan (CMCS) and 99.76% of toothpaste auxiliaries, by mass percentage.

[0025] As a preferred embodiment of the toothpaste of the present application, the toothpaste auxiliaries comprise abrasives, humectants, thickening agents, foaming agents, fragrances, taste modifiers, preservatives or water.

[0026] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.24-0.36% of the above-mentioned composition, 12-20% of a rubbing agent, 50-60% of a moisturizing agent, 0.8-1.5% of a thickening agent, 1.8-2.5% of a foaming agent, 0.8-1.5% of a fragrance, 0.18-0.3% of a taste modifier, 0.2-0.3% of a preservative, and the balance of water, by mass%.

[0027] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.04-0.06% of pyrroloquinoline quinone (PQQ), 0.2-0.3% of carboxymethyl chitosan (CMCS), 12-20% of a rubbing agent, 50-60% of a moisturizing agent, 0.8-1.5% of a thickening agent, 1.8-2.5% of a foaming agent, 0.8-1.5% of a fragrance, 0.18-0.3% of a taste modifier, 0.2-0.3% of a preservative, and the balance of water, by mass.

[0028] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.05-0.06% of pyrroloquinoline quinone (PQQ), 0.25-0.3% of carboxymethyl chitosan (CMCS), 12-20% of a rubbing agent, 50-60% of a moisturizing agent, 0.8-1.5% of a thickening agent, 1.8-2.5% of a foaming agent, 0.8-1.5% of a fragrance, 0.18-0.3% of a taste modifier, 0.2-0.3% of a preservative, and the balance of water, by mass.

[0029] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.05% of pyrroloquinoline quinone (PQQ), 0.25% of carboxymethyl chitosan (CMCS), 18% of a rubbing agent, 52% of a moisturizing agent, 0.8% of a thickening agent, 2.2% of a foaming agent, 1.2% of a fragrance, 0.25% of a taste modifier, 0.3% of a preservative, and the balance of water, by mass.

[0030] As a preferred embodiment of the toothpaste of the present application, the toothpaste comprises 0.04% of pyrroloquinoline quinone (PQQ), 0.2% of carboxymethyl chitosan (CMCS), 18% of a rubbing agent, 52% of a moisturizing agent, 0.8% of a thickening agent, 2.2% of a foaming agent, 1.2% of a fragrance, 0.25% of a taste modifier, 0.3% of a preservative, and the balance of water, by mass.

[0031] As a preferred embodiment of the toothpaste, the abrasive agent comprises hydrated silica or calcium hydrogen phosphate; the humectant comprises sorbitol, glycerol, polyethylene glycol-8 or propylene glycol; the thickening agent comprises sodium carboxymethyl cellulose, xanthan gum, hydroxypropyl guar gum, CHONDRUS CRISPUS or tamarind gum; the foaming agent comprises sodium lauryl sulfate, cocamidopropyl betaine or sodium lauryl sarcosinate; the fragrance is essence; the taste modifier comprises sodium saccharin, sucralose or stevia; and the preservative comprises sodium benzoate, hydroxybenzoate or hydroxypropylbenzoate.

[0032] In a fourth aspect, the application provides a preparation method of the toothpaste, comprising the following steps:

[0033] S1, mixing the abrasive agent, the foaming agent and the thickening agent in the toothpaste auxiliary materials to obtain a powder;

[0034] S2, mixing the water, the humectant, the taste modifier, the preservative, pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in the toothpaste auxiliary materials to obtain a premix;

[0035] S3, controlling the vacuum degree of the toothpaste preparation machine to be-0.02MPa to-0.04MPa, and sucking the premix of step S2 into the toothpaste preparation machine; starting the vacuum pump, the double stirring and the scraper of the toothpaste preparation machine, and stirring while pumping; controlling the vacuum degree to be-0.06MPa to-0.08MPa, and sucking the powder of step S1 into the toothpaste preparation machine; when the paste slowly descends and stabilizes without rising, keeping the vacuum degree above-0.090MPa, then stopping the vacuum pump, the double stirring and the scraper after stirring for 15-20 minutes; controlling the vacuum degree to be-0.02MPa to-0.04MPa, and sucking the fragrance into the toothpaste preparation machine; restarting the vacuum pump, the double stirring and the scraper, stirring for 10-15 minutes and then stopping the double stirring; keeping the vacuum degree above-0.090MPa, and stirring for 5-10 minutes by the scraper, thereby obtaining the toothpaste.

[0036] Compared with the prior art, the application has the following advantages:

[0037] The application applies pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in the toothpaste, which can promote the proliferation and repair of gingival fibroblasts (HGF) through daily use of the toothpaste, and effectively maintain the balance of oral microecology. The application plays a key role in improving the quality of life of individuals. The development of toothpaste products with health care functions can not only meet the growing diversified needs of consumers for oral health, but also promote technological innovation, enhance the market competitiveness of enterprises, and have a positive impact on the social economy. With the continuous enhancement of public health awareness, the health care toothpaste market shows great development potential. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure for toothpaste finished product of Example 5 of the present application. DETAILED DESCRIPTION

[0039] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further explained in combination with specific examples.

[0040] Other materials, reagents used in the examples, etc. can be obtained from commercial channels if not specially specified.

[0041] The CMCS used in the present application has a molecular weight of 5000-10000 Da and a carboxymethyl substitution degree of 0.8-1.2.

[0042] The serum source: sigma, high-sugar medium (DMEM) source: Gibco.

[0043] Examples 1-12 and Comparative Examples 1-3

[0044] A toothpaste comprising the composition 0.24-0.36% and toothpaste auxiliary materials 99.64-99.76% by mass percentage. The toothpaste auxiliary materials include abrasives, humectants, thickeners, foaming agents, fragrances, taste improvers, preservatives or water.

[0045] The toothpaste comprises the composition 0.24-0.36%, abrasives 12-20%, humectants 50-60%, thickeners 0.8-1.5%, foaming agents 1.8-2.5%, fragrances 0.8-1.5%, taste improvers 0.18-0.3%, preservatives 0.2-0.3% and water in residual amount by mass percentage.

[0046] The abrasives include hydrated silica or dibasic calcium phosphate; the humectants include sorbitol, glycerol, polyethylene glycol-8 or propylene glycol; the thickeners include sodium carboxymethyl cellulose, xanthan gum, hydroxypropyl guar gum, CHONDRUS CRISPUS or locust bean gum; the foaming agents include sodium lauryl sulfate, cocamidopropyl betaine or sodium lauroyl methylsulfate; the fragrances are essence; the taste improvers include sodium saccharin, sucralose or stevia; the preservatives include sodium benzoate, hydroxybenzoate or hydroxypropylbenzoate.

[0047] The composition components of the toothpaste of Examples 1-12 and Comparative Examples 1-3 are shown in Table 1 below,

[0048] Table 1

[0049]

[0050]

[0051] The preparation method of the toothpaste of Examples 1-12 and Comparative Examples 1-3 comprises the following steps:

[0052] (1) The abrasives, foaming agents and thickening agents in the toothpaste auxiliaries are added into the powder tank according to the formula amount to obtain a powder;

[0053] (2) The water, humectants, taste improvers, preservatives, and the composition of pyrroloquinoline quinone (PQQ) and carboxymethyl chitosan (CMCS) in the toothpaste auxiliaries are placed in a liquid premix barrel, and are uniformly dispersed to obtain a premix liquid;

[0054] (3) The vacuum degree of the toothpaste making machine is controlled to be -0.02 MPa to -0.04 MPa, and the prepared premix liquid is vacuumed into the toothpaste making machine; the vacuum pump, double stirring and scraper of the toothpaste making machine are started at room temperature, and the gas is extracted while stirring; the vacuum degree is controlled to be -0.06 MPa to -0.08 MPa, and the prepared powder is vacuumed into the toothpaste making machine; when the paste body begins to slowly descend and stabilizes without rising, the vacuum degree is maintained above -0.090 MPa, then the vacuum pump, double stirring and scraper are stopped after stirring for 15-20 minutes; the fragrance is sucked into the toothpaste making machine by controlling the vacuum degree to be -0.02 MPa to -0.04 MPa; the vacuum pump, double stirring and scraper are restarted, and the double stirring is stopped after stirring for 10-15 minutes; the vacuum degree is maintained above -0.090 MPa, and the scraper is stirred for 5-10 minutes to obtain the toothpaste.

[0055] The finished toothpaste of Example 5 is shown in the figure. Figure 1

[0056] Test Example 1: In vitro antibacterial effect test of toothpaste

[0057] According to WS / T 650-2019 "Evaluation method of antibacterial and bacteriostatic effect", the in vitro bacteriostatic experiment test is carried out. According to the carrier immersion quantitative bacteriostatic test method of WS / T 650-2019 "Evaluation method of antibacterial and bacteriostatic effect", the action time is 3 minutes, and the action concentration is: original sample. The test strains include: Streptococcus mutans ATCC 25175 and Porphyromonas gingivalis ATCC 33277.

[0058] The in vitro bacteriostatic effect of the toothpaste of Examples and comparative examples is shown in Table 2,

[0059] Table 2

[0060]

[0061] ​The results of Table 2 show that the antibacterial rates of Example 5 and Example 6 are higher than 50%, indicating that they have antibacterial effects. The sum of the antibacterial rates of Comparative Example 1 and Comparative Example 2 is less than that of Example 5, indicating that the combination of PQQ and CMCS has a synergistic effect.

[0062] PQQ can promote the oxidative stress of CMCS as an electron carrier, enhancing its ability to destroy the bacterial cell membrane; the cationic property of CMCS and the chelation of PQQ can form a complex antibacterial agent, targeting key bacterial metabolic enzymes (such as glucose-6-phosphate dehydrogenase), thereby exerting a synergistic antibacterial effect.

[0063] The inventors also prepared toothpaste by combining the composition of Example 5 with different contents of adjuvants, and the toothpaste comprises the following ingredients in mass percentage: pyrroloquinoline quinone (PQQ) 0.05%, carboxymethyl chitosan (CMCS) 0.25%, abrasives 12-20%, humectants 50-60%, thickening agents 0.8-1.5%, foaming agents 1.8-2.5%, fragrances 0.8-1.5%, taste improvers 0.18-0.3%, preservatives 0.2-0.3%, and water in the remainder. The prepared toothpaste all has antibacterial activity.

[0064] Test Example 2: Anti-inflammatory effect test of toothpaste

[0065] The toothpaste of Example 5 and Example 6 above were used as samples for the anti-inflammatory effect test.

[0066] Experimental animals: 24 SPF KM mice, male, 18-22 g.

[0067] Instruments and reagents: main instruments, METTLER TOLEDO AG204 electronic balance, 8 mm puncher; reagents, dimethylbenzene.

[0068] Test method: After quarantine, 36 SPF KM mice were randomly divided into 3 groups, 12 mice in each group, namely the test group of Example 5, the test group of Example 6, and the model control group. After the mice were quarantined and grouped, the left ear was not treated as the baseline value, and 30 μL of dimethylbenzene solution was applied to the inner and outer surfaces of the right ear. After 30 min, the model control group was not treated, and the test group of mice was applied with 0.1 g of the corresponding toothpaste on the right ear. After 1 h, the mice were sacrificed by cervical dislocation, the test substance was wiped off, and the ears were cut off. The ear pieces with a diameter of 8 mm (the same part of the left and right ears) were weighed and recorded. The weight difference of the left and right ears of the same animal was the degree of swelling. The swelling inhibition rate was calculated according to the degree of swelling. The ability to inhibit swelling indicates the anti-inflammatory effect.

[0069] Swelling degree = right ear piece weight - left ear piece weight.

[0070] Swelling inhibition rate (%) = (average degree of swelling of model control group - average degree of swelling of test group) / average degree of swelling of model control group x 100.

[0071] The experimental data are expressed as mean ± standard deviation Independent sample T test was performed using SPSS software.

[0072] The anti-inflammatory effect test results are shown in Table 3 below,

[0073] Table 3

[0074] Group Ear swelling degree (mg) Ear swelling inhibition rate (%) Model control group 22.36±2.78 - Example 5 13.02±3.75 41.77 Example 6 14.63±3.65 34.57

[0075] The results of Table 3 show that the ear swelling of the mice in the test group is reduced compared with the model control group, and the difference is statistically significant (P < 0.05). The toothpaste of Example 5 and Example 6 in the test group can inhibit the ear swelling of the mice. Compared with the model control group, the ear swelling inhibition rates of the mice treated with the toothpaste of Example 5 and Example 6 are 41.77% and 34.57%, respectively, indicating that the toothpaste formula containing PQQ and CMCS can significantly inhibit the ear swelling of the mice induced by xylene and has clear anti-inflammatory activity. The swelling inhibition rate of Example 5 is higher, and the anti-inflammatory effect is better.

[0076] Oral repair effect test of test example 3 toothpaste

[0077] 1. Method

[0078] The toothpaste of Example 4, Example 5, Example 6, Example 7, and Example 8 was used as a sample for the gingival fibroblast proliferation experiment. Gingival fibroblasts (HGF) are the main cell types of gingival connective tissue and periodontal membrane, which play an important role in the formation and regeneration of periodontal tissue, the maintenance of integrity, and the implementation of function. Gingival fibroblasts are abundant in source and have strong growth and self-reproduction ability. The content of serum has a significant effect on cell proliferation repair. 15% serum was used as a positive control. The cell proliferation rate of the sample group after administration was determined to evaluate the proliferation effect of the sample on cells.

[0079] Instruments: inverted microscope, enzyme marker, carbon dioxide incubator, centrifuge, cell counter, biological safety cabinet.

[0080] Experimental materials: 2% serum medium (2% serum (FBS) + 98% high-sugar medium (DMEM)), 15% serum medium (15% FBS + 85% DMEM), thiazolyl blue (MTT) working solution material (dilute 5 mg / mL MTT stock solution with complete culture medium to 1 mg / mL MTT working solution, prepare and use immediately), gingival fibroblasts (HGF, cells are derived from Beijing Bolikangmu Biotechnology Co., Ltd., and the cell proliferation test is 05 generation)

[0081] The test groups are shown in Table 4 below,

[0082] Table 4

[0083]

[0084] Experimental procedures:

[0085] (1) Cell resuscitation and subculture;

[0086] (2) Plating

[0087] Select cells in the logarithmic growth phase, collect the cells after trypsin digestion, prepare a cell suspension, count using a cell counter, inoculate the cell suspension into 3 96-well plates, and name them plate 1, plate 2 and plate 3, respectively. The cells should be cultured for 24 h to reach a confluence of 20%-30%, with a liquid volume of 100 μL per well. After inoculation, incubate in a 37°C, 5% CO2 incubator for 24±2 h.

[0088] (3) Drug administration

[0089] Discard the culture medium in the 96-well cell culture plate, administer the drug according to the test groups, 100 μL per well, and incubate in a 37°C, 5% CO2 incubator for 24-48 h.

[0090] (4) MTT test

[0091] Plate 1 was removed after 24 h of plating, and the MTT test was performed according to the cell toxicity experiment procedure of the MTT method to determine the absorbance of each group. Plates 2 and 3 were removed after 24 h and 48 h of drug administration, respectively, and the MTT test was performed according to the cell toxicity experiment procedure of the MTT method to determine the absorbance of each group. The cell proliferation rate was calculated according to the absorbance,

[0092] Cell proliferation rate (%) = experimental group OD / blank control group OD × 100.

[0093] The test results show that the coefficient of variation CV value of the OD value of each group is less than 20%, and the test system is effective.

[0094] 2. Results

[0095] The effect of toothpaste on cell proliferation is shown in Table 5 below,

[0096] Table 5

[0097] Experimental grouping 0h proliferation rate (Mean ± SD) 24h proliferation rate (Mean ± SD) 48h proliferation rate (Mean ± SD) Blank control group 100.00±1.10％ 98.06±1.22％ 97.08±13.65％ Positive control group 100.00±1.10％ 171.52±2.17％ 242.11±2.01％ Example 4 100.00±1.10％ 169.05±1.09％ 156.21±7.07％ Example 5 100.00±1.10％ 161.29±4.82％ 149.09±5.87％ Example 6 100.00±1.10％ 36.40±0.95％ 36.95±1.39％ Example 7 100.00±1.10％ 37.61±2.13％ 36.82±1.16％ Example 8 100.00±1.10％ 36.40±4.50％ 36.42±0.83％

[0098] The results of Table 5 show that the gingival fibroblasts (HGF) in the positive control group and the blank control group were significantly increased in cell proliferation rate at 24h and 48h. The cell proliferation rates of Examples 4 and 5 were significantly improved at 24h and 48h, close to the positive control group, showing a stable cell proliferation promoting effect, indicating that the toothpaste containing PQQ and CMCS in the concentration range of Examples 4 and 5 has the effect of promoting oral repair. The toothpaste of Example 4 has a better effect of promoting oral repair.

[0099] Test Example 4 Oral Microecological Evaluation of Toothpaste

[0100] 1. Method

[0101] The toothpaste of Example 5 was used as the test sample, the ordinary commercially available toothpaste was used as the negative control group, and the 0.12% chlorhexidine gargle was used as the positive control group for oral microecological testing.

[0102] Volunteers were recruited and divided into three groups, each group with ≥30 people.

[0103] Volunteer recruitment requirements:

[0104] Age range: Choose adults aged 18-65 years old. Non-smokers, or those who have not smoked for at least 6 months in the past.

[0105] General health: Volunteers should be in good health, without systemic diseases such as diabetes, cardiovascular disease, immune system disease, etc. Good periodontal condition, no obvious gingival inflammation (such as gingival swelling, bleeding, etc.), periodontitis (such as periodontal pocket formation, alveolar bone resorption, etc.).

[0106] Drug use: No use of antibiotics, antifungal drugs, immunosuppressants, etc. in the recent period (such as within the past 1 month).

[0107] Allergy history: No known history of allergy to toothpaste ingredients, chlorhexidine or other oral care products.

[0108] Oral mucosa health: No lesions such as ulcers, wounds, leukoplakia, erythema on the oral mucosa.

[0109] Denture condition: If wearing dentures, should be fixed dentures and wearing time more than 3 months, no denture related discomfort or inflammation.

[0110] Alcohol consumption: No history of heavy drinking in the recent period (such as within the past 1 week).

[0111] Oral hygiene habits: Volunteers should maintain relatively stable oral hygiene habits, such as the number of times of brushing teeth per day (recommended 2-3 times), brushing method (such as the Bass brushing method), etc. During the test period, volunteers were required to clean their mouths according to their usual habits, but were required to avoid using other oral care products, such as mouthwash, dental floss, etc. (except for the toothpaste specified in the test).

[0112] Volunteers used three groups of toothpaste for 0 days (baseline), 7 days (acute phase), and 28 days (sub-stable phase), respectively, and then collected oral plaque for microbial community diversity analysis.

[0113] Sample collection: Sterile curettes were used to collect supragingival plaque and immediately freeze at -80°C. Subgingival plaque was centrifuged within 30 minutes. Saliva collectors were used to collect non-stimulating whole saliva (2 mL / tube, avoiding food residue contamination). The samples were stored at 4°C for ≤24 hours and at -80°C for long-term storage.

[0114] DNA extraction: The QIAamp DNA Microbiome Kit was used for mechanical crushing (bead mill at 30 Hz for 5 min) and enzymatic treatment (lysozyme at 5 mg / mL, incubated at 37°C for 1 h) according to the manufacturer's instructions to ensure a bacterial lysis rate of ≥90% and a host DNA contamination rate of ≤5%.

[0115] Amplified sequencing: PCR amplification was performed using 16S rRNA V3-V4 region primers (primer 341F: 5'-CCTAC-GGG NGGCWGCAG-3', primer 805R: 5'-GACTACH-VGGGTATCTAATCC-3') with an annealing temperature of 55°C and 25 cycles. The sequencing depth was ≥10,000 reads / sample, and the coverage rate of core flora (Firmicutes, Proteobacteria, Bacteroidetes) was ≥95%. The sequencing platform was Illumina NovaSeq 6000 (PE250 mode), and the sequencing depth was ≥50,000 reads / sample.

[0116] Community diversity analysis (delegated to a third detection agency):

[0117] (1) Alpha diversity (Shannon index)

[0118] Data preprocessing: The raw data obtained by sequencing was subjected to quality control and filtering to remove low-quality sequences and chimeras, resulting in valid sequence data.

[0119] OTU clustering: QIIME 2 software was used to cluster valid sequences according to a similarity level of 97%, generating operational taxonomic units (OTUs).

[0120] Calculation of Shannon index: Based on the OTU clustering results, the vegan package of R language was used to calculate the Shannon index of each sample. This index considers both the richness and evenness of the flora, and a higher value indicates higher flora diversity.

[0121] (2) Beta diversity (PCoA analysis)

[0122] Distance matrix calculation: Bray-Curtis distance algorithm was used to calculate the difference in microbial community structure between samples, and a distance matrix was generated.

[0123] PCoA analysis: The vegan package and ggplot2 package of R language were used to perform principal coordinate analysis (PCoA) on the distance matrix, and the high-dimensional microbial community structure data was reduced to two-dimensional or three-dimensional space to intuitively show the similarity and difference of microbial community structure between different samples.

[0124] (3) Dynamic changes of key microbial communities

[0125] Species annotation: The OTU representative sequence was aligned with the database (SILVA database) to determine the microbial species information corresponding to each OTU.

[0126] Relative abundance calculation: The number of sequences of each key microbial community in each sample was counted, and the relative abundance in the total sequences was calculated.

[0127] Dynamic change analysis: The relative abundance changes of each key microbial community at different time points (0 days, 7 days, 28 days) were compared, and the statistical method (t-test or analysis of variance) was used to analyze the significance of the difference.

[0128] 2、Results

[0129] (1) The results of α diversity analysis are shown in Table 6,

[0130] Table 6

[0131]

[0132] The results in Table 6 show that the Shannon index of the toothpaste of Example 5 is significantly higher than that of the negative control group at 7 days (P<0.05), indicating an increase in microbial diversity. The positive control group (chlorhexidine gargle) significantly reduces the microbial diversity (P<0.01), and maintains a low level at 28 days. It shows that the toothpaste containing PQQ and CMCS of the present application can increase the diversity of oral microbial community.

[0133] (2) The results of β diversity analysis show that the similarity of microbial community structure of the toothpaste of Example 5 to the negative control group is 85% at 28 days, but the proportion of beneficial bacteria (such as Streptococcus salivarius and Lactobacillus rhamnosus) is significantly higher than that of the negative control group (P<0.01). The microbial community structure of the positive control group is significantly different from the baseline (ANOSIM R=0.65, P<0.001), indicating that chlorhexidine leads to the simplification of microbial community structure. It shows that the toothpaste containing PQQ and CMCS of the present application can increase the diversity of oral microbial community and increase the number of beneficial bacteria.

[0134] (3) Key microbiota dynamic change analysis results show that for supragingival plaque, the toothpaste of Example 5, the proportion of Streptococcus salivarius (beneficial bacteria) increased from 12% ± 3% at 0 days baseline to 24% ± 4% at 28 days (P < 0.01). The proportion of Porphyromonas gingivalis (pathogenic bacteria) decreased from 8% ± 2% at 0 days baseline to 2% ± 1% at 28 days (P < 0.01). In the positive control group, the proportion of Porphyromonas gingivalis decreased to 0.5% ± 0.2%, but the proportion of Streptococcus also decreased significantly (from 15% ± 3% to 5% ± 1%, P < 0.01).

[0135] For subgingival plaque, the toothpaste of Example 5, the proportion of Fusobacterium nucleatum (periodontitis-related bacteria) decreased from 6% ± 2% at baseline to 1% ± 0.5% at 28 days (P < 0.01); the proportion of Veillonella (commensal bacteria) increased from 5% ± 1% at baseline to 12% ± 2% at 28 days (P < 0.01).

[0136] For saliva, the toothpaste of Example 5, the proportion of Neisseria (involved in nitrogen cycle) increased from 7% ± 2% at baseline to 15% ± 3% at 28 days (P < 0.01); the proportion of Prevotella (potential pathogenic bacteria) decreased from 10% ± 3% at baseline to 4% ± 1% at 28 days (P < 0.01).

[0137] In summary, the toothpaste containing PQQ and CMCS of the present application is superior to chlorhexidine gargle in regulating bacterial diversity, inhibiting pathogenic bacteria and promoting the proliferation of beneficial bacteria, and has no ecological damage, and is suitable for long-term use.

[0138] Stability test of toothpaste of Test Example 5

[0139] The prepared toothpaste product was placed in an electric thermostatic drying oven at 45°C, and after 3 months, the sample stored at room temperature was taken out and compared. The changes in appearance, fragrance, pH value, etc. of the high-temperature treated sample were observed to infer the stability of the toothpaste within the 3-year shelf life.

[0140] The test results show that the toothpaste prepared by the present application has good stability. The stability results of Example 5 are shown in Table 7 as follows,

[0141] Table 7

[0142]

[0143] The toothpaste containing PQQ and CMCS significantly improves the oral microecological structure through the double mechanism of selectively inhibiting pathogenic bacteria and directionally enriching probiotics / syntrophs, optimizes the function of the flora while improving the diversity of the flora, and has the effects of bacteriostasis, anti-inflammation and ecological restoration. The clinical value thereof is reflected in the prevention and adjuvant treatment of periodontitis, dental caries and halitosis, and the safety and compliance thereof are significantly better than those of traditional antibacterial agents (such as chlorhexidine), and the toothpaste is expected to become a core component of a new generation of oral health products.

[0144] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A composition having antibacterial, anti-inflammatory and / or oral repair efficacy, characterized in that, 0.04-0.06 parts by weight of pyrrolquinoline quinone and 0.2-0.3 parts by weight of carboxymethyl chitosan.

2. The composition of claim 1, wherein 0.05-0.06 parts by weight of pyrrolquinoline quinone and 0.25-0.3 parts by weight of carboxymethyl chitosan.

3. The composition of claim 1, wherein 0.04-0.05 parts by weight of pyrrolquinoline quinone and 0.2-0.25 parts by weight of carboxymethyl chitosan.

4. Use of the composition of any one of claims 1-3 in the preparation of toothpaste.

5. A toothpaste, characterized by 0.24-0.36% by mass of the composition of any one of claims 1-3 and 99.64-99.76% by mass of toothpaste auxiliary materials.

6. The toothpaste according to claim 5, wherein 0.04-0.06% by mass of pyrrolquinoline quinone, 0.2-0.3% by mass of carboxymethyl chitosan, and 99.64-99.76% by mass of toothpaste auxiliary materials.

7. A toothpaste according to claim 5 or 6, wherein the amount of the sodium salt of the anionic surfactant is 0.1 to 0.5% by weight. The toothpaste auxiliary materials include abrasives, humectants, thickening agents, foaming agents, fragrances, taste improvers, preservatives, or water.

8. The toothpaste according to claim 7, wherein 0.24-0.36% by mass of the composition of any one of claims 1-3, 12-20% by mass of abrasives, 50-60% by mass of humectants, 0.8-1.5% by mass of thickening agents, 1.8-2.5% by mass of foaming agents, 0.8-1.5% by mass of fragrances, 0.18-0.3% by mass of taste improvers, 0.2-0.3% by mass of preservatives, and the balance of water.

9. A toothpaste according to claim 7 or 8, wherein the amount of the sodium salt of the anionic surfactant is 0.1 to 0.5% by weight. The abrasives include hydrated silica or dibasic calcium phosphate; and / or, the humectants include sorbitol, glycerol, polyethylene glycol-8, or propylene glycol; and / or, the thickening agents include sodium carboxymethyl cellulose, xanthan gum, hydroxypropyl guar gum, chondrus crispus, or tragacanth gum; and / or, the foaming agents include sodium lauryl sulfate, cocamidopropyl betaine, or sodium lauroyl sarcosinate; and / or, the fragrances are essences; and / or, the taste improvers include sodium saccharin, sucralose, or stevia; and / or, the preservatives include sodium benzoate, methylparaben, or propylparaben.

10. A process for the preparation of a toothpaste according to claim 8 or 9, characterised in that, The method comprises the following steps: S1, mixing the abrasives, foaming agents, and thickening agents in the toothpaste auxiliary materials to obtain a powder; S2, mixing the water, humectants, taste improvers, preservatives, pyrrolquinoline quinone, and carboxymethyl chitosan in the toothpaste auxiliary materials to obtain a premix; S3, control the vacuum degree of the paste making machine to be -0.02MPa to -0.04MPa, and suck the premix liquid of step S2 into the paste making machine; start the vacuum pump, double stirring and scraper of the paste making machine, and stir while pumping; control the vacuum degree to be -0.06MPa to -0.08MPa, and suck the powder of step S1 into the paste making machine; when the paste body slowly drops and is stable without rising, keep the vacuum degree above -0.090MPa, then stop the vacuum pump, double stirring and scraper after stirring for 15 to 20 minutes; control the vacuum degree to be -0.02MPa to -0.04MPa, and suck the fragrance into the paste making machine; restart the vacuum pump, double stirring and scraper, stir for 10 to 15 minutes, stop the double stirring; keep the vacuum degree above -0.090MPa, and stir for 5 to 10 minutes by the scraper, thus the toothpaste is obtained.

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