An oral care composition and a preparation method thereof

Abstract

The present invention belongs to the technical field of oral care. More specifically, it relates to an oral care composition and a preparation method thereof. The oral care composition contains an enzymolysis product of royal jelly. The present invention for the first time discovers that the product with a molecular weight between 3 and 5 kDa isolated from the enzymolysis product of royal jelly enzymolyzed under specific conditions has a significant effect on promoting the proliferation and migration of oral mucosal epithelial cells, which suggests that this product may have a repairing effect on oral mucosal damage and can be applied to the treatment of oral mucosal diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of oral care. More specifically, it relates to an oral care composition and a preparation method thereof. Background Art

[0002] Royal jelly, also known as: Wang jelly, bee royal jelly, royal jelly, bee milk, is a milky white gelatinous substance secreted by the hypopharyngeal glands of worker bees of the bee family Apis cerana Fabricius and Apis mellifera L. According to research: Abundant superoxide dismutase (SOD), transaminase, cholinesterase, antibacterial peptides and various major royal jelly proteins (MRJPs) are important bases for royal jelly to achieve functions of anti-aging, anti-tumor, antibacterial and immune regulation.

[0003] Oral mucosal diseases are a general term for diseases occurring in oral mucosa and soft tissues. Common oral mucosal diseases include oral candidiasis, oral lichen planus, recurrent aphthous ulcer of the mouth, etc. Common symptoms are mucosal burning pain, dry mouth, taste abnormality, etc. The treatment of oral mucosal diseases is based on the principles of anti-inflammatory, anti-infection, pain relief and promoting healing. Currently, there is no research on using enzymolysis products of royal jelly for the treatment of oral mucosal diseases. Summary of the Invention

[0004] The object of the present invention is to provide the use of enzymolysis products of royal jelly in the preparation of an oral mucosal repair composition.

[0005] Another object of the present invention is to provide an oral care composition.

[0006] The inventor of the present invention conducted repeated and in-depth research to achieve the above object, and found that the product with a molecular weight between 3 and 5 kDa isolated from the enzymolysis products of royal jelly enzymolyzed under specific conditions showed the effect of promoting the proliferation and migration of oral mucosal epithelial cells within a certain concentration range, suggesting that this product may have a repairing effect on oral mucosal damage and can be applied to the prevention and treatment of oral mucosal diseases such as oral candidiasis, oral lichen planus, recurrent aphthous ulcer of the mouth, etc.

[0007] This effect is related to the type of enzyme and the molecular weight of the product. The enzymolysis products obtained by different enzyme combinations show different physiological activities. By comparing different enzyme combinations, it is found that the product with a molecular weight of <5 kDa isolated from the enzymolysis products of bromelain + proteinase K shows the strongest proliferation-promoting and migration-promoting effects, while the enzymolysis products of papain + proteinase K in each molecular weight segment do not show the same proliferation-promoting activity, and the proliferation-promoting activity of the enzymolysis products of bromelain + pepsin is significantly lower than that of the bromelain + proteinase K combination.

[0008] Secondly, there are also significant differences in the activities exhibited by products with different molecular weights isolated from the products obtained by enzymatic hydrolysis with the same enzyme combination. For example, the proliferation-promoting activity of the royal jelly enzymatic hydrolysis product of bromelain + proteinase K with a molecular weight of 3 - 5 kDa is significantly greater than that with a molecular weight < 3 kDa and extremely significantly greater than that with a molecular weight > 5 kDa, and the differences between the two are statistically significant.

[0009] The above object of the present invention is achieved by the following technical solutions:

[0010] On the one hand, the present invention provides the use of a royal jelly enzymatic hydrolysis product in the preparation of an oral mucosa repair composition, and the royal jelly enzymatic hydrolysis product is prepared by the following steps:

[0011] S1. Add royal jelly to deionized water according to a certain ratio, stir well, let stand at 4°C for 30 - 60 min, centrifuge, and take the supernatant.

[0012] S2. Add bromelain to the supernatant, mix well, and carry out enzymatic hydrolysis for 2 - 6 h; then add proteinase K, mix well, and carry out enzymatic hydrolysis for 1 - 3 h.

[0013] S3. After the enzymatic hydrolysis is completed, inactivate, centrifuge, filter the supernatant with an ultrafiltration membrane, and dry to obtain the royal jelly enzymatic hydrolysis product.

[0014] As one embodiment of the present invention, in step S2, the conditions for bromelain enzymatic hydrolysis are: the enzyme addition amount is 300 - 500 U / g, the reaction temperature is 45 - 65°C, and the pH is 6.0 - 8.0.

[0015] As one embodiment of the present invention, in step S2, the conditions for proteinase K enzymatic hydrolysis are: the enzyme addition amount is 300 - 500 U / g, the reaction temperature is 50 - 60°C, and the pH is 7.5 - 11.0.

[0016] As one embodiment of the present invention, the filtration is carried out using ultrafiltration membranes with molecular weights of 3 kDa and 5 kDa.

[0017] As one embodiment of the present invention, the molecular weight of the royal jelly enzymatic hydrolysis product is 3 - 5 kDa.

[0018] As one embodiment of the present invention, the drying method in step S3 includes spray drying, freeze drying, or ventilation drying.

[0019] On the other hand, the present invention provides an oral care composition, and the composition contains the above-mentioned royal jelly enzymatic hydrolysis product.

[0020] The above-mentioned oral care composition, in addition to the active ingredients described above, can also be appropriately formulated with ingredients commonly used in the art as needed. Secondly, the oral care composition includes toothpaste, mouthwash, etc., and preferably is prepared as toothpaste. In addition to the necessary active ingredients, it may also contain additives commonly used in the art, such as: humectants, preservatives, colorants, taste modifiers, thickeners, foaming agents, abrasives, and edible flavors, etc.

[0021] Among them, the main function of the humectant is to keep the toothpaste paste moist and maintain a certain amount of moisture. When the paste is exposed to the air, it can effectively prevent the evaporation and loss of moisture in the paste, and also prevent the toothpaste at the nozzle from drying out easily during the storage of the toothpaste product, making it easy to extrude and convenient for consumers to use; at the same time, it is also beneficial to improve and maintain the rheology of the paste, facilitating filling during production; reducing the freezing point; and improving the stability of the toothpaste. Examples of humectants that can be used include: polyethylene glycol, sorbitol, glycerol, and propylene glycol, etc.

[0022] As the main raw material of toothpaste, abrasives that can be listed include: natural calcium carbonate, precipitated silica, dicalcium hydrogen phosphate dihydrate, aluminum hydroxide, light calcium carbonate, anhydrous calcium hydrogen phosphate, etc., and preferably calcium oxide.

[0023] The function of the foaming agent is to provide excellent emulsifying properties and good foaming properties. Examples of foaming agents that can be listed include: sodium lauryl sulfate, sodium lauryl sulfate, cocamidopropyl betaine, alkyl polyglycoside (APG) classes, and polyglycerol esters.

[0024] The thickener is preferably a natural thickener, such as: alginic acid (salt), starch, gum arabic, guar gum, carrageenan, pectin, and agar, etc.

[0025] The taste modifier is preferably a natural sweetener, such as xylitol, stevioside, mogroside, glycyrrhizin, rubusoside, thaumatin, and honey, etc.

[0026] The preservative can be a natural preservative or a chemical preservative. Among them, natural preservatives such as: magnolia officinalis extract, grapefruit extract, peony root extract, chitosan, and lysozyme, etc. Chemical preservatives such as: benzoic acid and its salts, sorbic acid and its salts, and parabens, etc.

[0027] The present invention has the following beneficial effects:

[0028] The present invention for the first time discovers that the product with a molecular weight between 3 and 5 kDa isolated from the enzymolysis product of royal jelly enzymolyzed under specific conditions has a significant effect of promoting the proliferation and migration of oral mucosal epithelial cells, which suggests that this product has a repair effect on oral mucosal damage and can be applied to the treatment of oral mucosal diseases. Description of the Drawings

[0029] Figure 1 Results of Transwell migration experiments after treatment with A2, the enzymatic hydrolysis product of royal jelly at different concentrations;

[0030] Among them, Figure 1 ① is the control group, Figure 1 ② - ④ are the treatment groups with A2, the enzymatic hydrolysis product of royal jelly at 5mg / mL, 10mg / mL, and 15mg / mL respectively.

[0031] Figure 2 Statistical chart of the number of cell migrations of human oral mucosal epithelial cells after treatment with A2, the enzymatic hydrolysis product of royal jelly at different concentrations;

[0032] Among them, compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001. Specific implementation mode

[0033] The present invention can be further described through the following examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0034] Example 1. Preparation of the enzymatic hydrolysis product of royal jelly

[0035] S1. Add royal jelly to deionized water at a ratio of 1:10, stir well, let stand at 4°C for 45 min, centrifuge at 4000g for 10 min, and take the supernatant;

[0036] S2. Add bromelain to the supernatant at an enzyme addition amount of 400U / g, mix well, and enzymatically hydrolyze at 50°C and pH 7.0 for 4 h; then add proteinase K at 300U / g, mix well, and enzymatically hydrolyze at 55°C and pH 7.5 for 2.5 h;

[0037] S3. After the enzymatic hydrolysis is completed, inactivate the enzyme at 100°C for 5 min, centrifuge, and filter the supernatant through ultrafiltration membranes with molecular weights of 5 kDa and 3 kDa in sequence. After freeze-drying, the enzymatic hydrolysis product A1 of royal jelly with a molecular weight < 3 kDa, the enzymatic hydrolysis product A2 of royal jelly with a molecular weight of 3 kDa - 5 kDa, and the enzymatic hydrolysis product A3 of royal jelly with a molecular weight > 5 kDa are obtained respectively.

[0038] Comparative Example 1: The difference from Example 1 is that papain is used to replace bromelain, and the enzymatic hydrolysis conditions are as follows: the enzyme addition amount is 400 U / g, the temperature is 55 °C, and the pH is 7.5. The other parameters are the same as those in Example 1. The enzymatic hydrolysis products of royal jelly with a molecular weight < 3 kDa, B1, the enzymatic hydrolysis products of royal jelly with a molecular weight of 3 kDa - 5 kDa, B2, and the enzymatic hydrolysis products of royal jelly with a molecular weight > 5 kDa, B3, are obtained respectively.

[0039] Comparative Example 2: The difference from Example 1 is that pepsin is used to replace proteinase K, and the enzymatic hydrolysis conditions are as follows: the enzyme addition amount is 300 U / g, the temperature is 37 °C, and the pH is 2.0. The other parameters are the same as those in Example 1. The enzymatic hydrolysis products of royal jelly with a molecular weight < 3 kDa, C1, the enzymatic hydrolysis products of royal jelly with a molecular weight of 3 kDa - 5 kDa, C2, and the enzymatic hydrolysis products of royal jelly with a molecular weight > 5 kDa, C3, are obtained respectively.

[0040] Experimental Example 1: Proliferation promotion test of oral mucosal epithelial cells

[0041] Human oral mucosal epithelial cells in the third-generation logarithmic growth phase are taken and prepared into a cell suspension with a density of 1×10 6 cells / mL using a complete medium for human oral mucosal epithelial cells (purchased from Wuhan Punosai Life Science Co., Ltd., product number CM-H203). They are inoculated into a 96-well plate at 100 μL per well, with 8 wells in each group. After culturing for 24 h, the enzymatic hydrolysis products of royal jelly A1 - A3, B1 - B3, and C1 - C3 prepared above are respectively formulated into test solutions with a concentration of 10 mg / mL using the complete medium, and 100 μL of the test solution is added to each well of the 96-well plate. After culturing for 24 h under the conditions of 37 °C and 5% CO 2 , the culture solution is aspirated and discarded. According to the instructions of the CCK-8 kit, CCK-8 solution is added to each well, and incubation is continued under the conditions of 37 °C and 5% CO 2 for 4 h. The absorbance (A) value is measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader. The experiment is repeated 3 times, and the average value is taken to calculate the cell proliferation rate. The results are shown in Table 1 below.

[0042] Cell proliferation rate = (absorbance value of the experimental group - absorbance value of the blank control) / (absorbance value of the control group - absorbance value of the blank control) × 100%

[0043] Among them, the experimental group is the cells treated with the enzymatic hydrolysis products of royal jelly, the control group is the untreated cells, and the blank control group is the medium without cells.

[0044] Table 1. Effects of different enzymatic hydrolysis products of royal jelly on the proliferation of human oral mucosal epithelial cells

[0045] Grouping Proliferation rate (%) A1 Enzymatic Hydrolysate of Royal Jelly <![CDATA[148.62±5.21 ** > A2 Enzymatic Hydrolysate of Royal Jelly 175.94±10.35 A3 Enzymatic Hydrolysate of Royal Jelly <![CDATA[92.15±2.66 *** > B1 Enzymatic Hydrolysate of Royal Jelly <![CDATA[102.35±9.11 *** > B2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[92.44±6.07 *** > B3 Enzymatic Hydrolysate of Royal Jelly <![CDATA[83.52±3.48 *** > C1 Enzymatic Hydrolysate of Royal Jelly <![CDATA[136.89±8.24 ** > C2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[115.33±7.51 *** > C3 Enzymatic Hydrolysate of Royal Jelly <![CDATA[90.34±5.26 *** >

[0046] Note: Compared with the A2 group of the enzymatic hydrolysate of royal jelly, ** P < 0.01, *** P < 0.001.

[0047] When the cell proliferation rate approaches 100%, it indicates that the number of cells in the experimental group is equivalent to that in the control group; when the cell proliferation rate is less than 100%, it indicates that the number of cells in the experimental group is less than that in the control group; and when the cell proliferation rate is greater than 100%, it indicates that the number of cells in the experimental group is more than that in the control group.

[0048] As can be seen from Table 1 above, the enzymatic hydrolysates of royal jelly obtained with different combinations of composite enzymes showed different effects on human oral mucosal epithelial cells, some showing a promoting effect and some showing an inhibitory effect. This suggests that the types of enzymes and the molecular weight of the products during the hydrolysis process play a decisive role in the physiological activity of the enzymatic hydrolysates of royal jelly. Among them, the product with a molecular weight < 5 kDa obtained by hydrolysis with bromelain + proteinase K has a more obvious effect on the proliferation of human oral mucosal epithelial cells. Among them, the enzymatic hydrolysate A2 of royal jelly with a molecular weight of 3 - 5 kDa has the strongest promoting effect on the proliferation of human oral mucosal epithelial cells, with a proliferation rate reaching 175.94%. Followed by the enzymatic hydrolysate A1 of royal jelly with a molecular weight < 3 kDa, with a proliferation rate of 148.62%.

[0049] Experimental Example 2: Effects of Different Concentrations of Enzymatic Hydrolysate A2 of Royal Jelly on the Proliferation of Human Oral Mucosal Epithelial Cells

[0050] The enzymatic hydrolysate A2 of royal jelly was prepared into test solutions with different concentrations using a complete medium, and its effect on the proliferation activity of human oral mucosal epithelial cells was investigated by referring to the method of Experimental Example 1. The results are shown in Table 2 below.

[0051] Table 2 Effects of Different Concentrations of Enzymatic Hydrolysate A2 of Royal Jelly on the Proliferation of Human Oral Mucosal Epithelial Cells

[0052]

[0053] Grouping Proliferation rate (%) 1mg / mL A2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[118.46±3.81 ** > 5mg / mL A2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[156.33±10.54 * > 10mg / mL A2 Enzymatic Hydrolysate of Royal Jelly 182.31±12.06 15mg / mL A2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[127.06±9.42 ** > 20mg / mL A2 Enzymatic Hydrolysate of Royal Jelly <![CDATA[109.17±8.53 ** >

[0054] Note: Compared with the enzymatic hydrolysate A2 of royal jelly at 10 mg / mL, * P < 0.05, ** P < 0.01.

[0055] As can be seen from Table 2, the enzymatic hydrolysate A2 of royal jelly has a promoting effect on the proliferation of human oral mucosal epithelial cells in the range of 1 - 20 mg / mL, and promotes the proliferation of oral mucosal epithelial cells in a dose-dependent manner in the range of 1 - 10 mg / mL. The peak appears at 10 mg / mL, and at this time, the proliferation rate of human oral mucosal epithelial cells reaches 182.31%.

[0056] Experimental Example 3: Detection of cell migration using Transwell chambers

[0057] Resuspend the cultured human oral mucosal epithelial cells in serum-free complete medium for human oral mucosal epithelial cells for 24 h, digest the cells, wash them twice with PBS, then resuspend the cells in complete medium for human oral mucosal epithelial cells to prepare a cell suspension, and adjust the cell density to 5×10 5 cells / mL. Place the Transwell chamber in a 24-well plate, add 100 μL of the cell suspension to the Transwell chamber, and add the enzymatically hydrolyzed product A2 of royal jelly to the chamber at a ratio of test solution:cell suspension of 1:1. Incubate in a 37 °C, 5% CO 2 cell incubator for 24 h. Take out the Transwell chamber, remove the culture medium, and gently wipe the cells in the chamber with a cotton swab or cotton moistened with PBS. Add 600 μL of 4% paraformaldehyde fixative to the 24-well plate, place the chamber in the 24-well plate for fixation for 30 min, then discard the fixative, wash twice with PBS. Add the crystal violet working solution to the 24-well plate, place the chamber in for staining for 10 min, take out the chamber, wash the inside and outside of the chamber 3 times with PBS, air dry, place the Transwell chamber under a microscope for photography, and select 3 - 5 fields of view to count the number of migrated cells. The results are as follows Figure 1 shown.

[0058] From Figure 1 and Figure 2 it can be seen that compared with the control group, the number of migrated cells was significantly increased after culturing with the enzymatically hydrolyzed product A2 of royal jelly at a concentration of 5 - 15 mg / mL. Among them, the promoting effects were most obvious at concentrations of 5 mg / mL and 10 mg / mL, showing a highly significant difference compared with the control group.

[0059] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of royal jelly enzymatic hydrolysate in preparing an oral mucosa repair composition, characterized in that: The molecular weight of the royal jelly enzymatic hydrolysate is 3-5 kDa, and the royal jelly enzymatic hydrolysate is prepared by the following steps: S1. Add royal jelly to deionized water in a certain proportion, stir well, let stand at 4°C for 30-60 minutes, centrifuge, and take the supernatant; S2. Add bromelain to the supernatant, mix thoroughly, and then enzymolyze for 2 to 6 hours; then add proteinase K, mix thoroughly, and then enzymolyze for 1 to 3 hours; S3. After the enzymatic hydrolysis is completed, the product is inactivated and centrifuged. The supernatant is filtered using an ultrafiltration membrane and dried to obtain the royal jelly enzymatic hydrolysis product. The filtration is performed using 3kDa and 5kDa ultrafiltration membranes.

2. The use according to claim 1, characterized in that In step S1, the ratio of royal jelly to deionized water is 1:9-15; and the centrifugation conditions are: 3000-6000 g, 10-25 min.

3. The use according to claim 1, characterized in that In step S2, the conditions for enzymatic hydrolysis of bromelain are: enzyme addition amount is 300-500 U / g, reaction temperature is 45-65° C., and pH is 6.0-8.

0.

4. The use according to claim 1, characterized in that In step S2, the conditions for the proteinase K enzymolysis are: the enzyme addition amount is 300-500 U / g, the reaction temperature is 50-60° C., and the pH is 7.5-11.

0.

5. The use according to claim 1, characterized in that: The drying method in step S3 includes spray drying, freeze drying or ventilation drying.

6. An oral care composition, characterized in that The composition comprises the royal jelly enzymatic hydrolysate according to any one of claims 1 to 5.

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