Use of a mussel-like mucin and intermediates thereof having long-lasting moisturizing efficacy

Abstract

The application provides a protein intermediate and the use of the mussel adhesive protein obtained by oxidizing the protein intermediate by tyrosinase in a moisturizer. The protein intermediate and the mussel adhesive protein provided by the application have excellent long-acting moisturizing effect, and have wide application prospects as moisturizers.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of protein, and particularly relates to the use of a mussel-like mucin with long-acting moisturizing effect and intermediates thereof. BACKGROUND

[0002] The skin of human has a natural moisturizing system, which is composed of water, natural moisturizing factor (NMF) and lipid, and plays an important role in maintaining skin elasticity. The molecular structure of NMF is rich in hydroxyl groups, which can bind water molecules, and the lipid thinly covers the surface of the skin to prevent water molecules from escaping into the environment and prevent water evaporation. The content of the three components of the natural moisturizing system and their balance have a great impact on the skin. In the dry air of autumn and winter, the physiological function of the skin will change, the water evaporation will become faster, the blood circulation will slow down, and the excretion of sweat and sebum will decrease, so the skin is prone to dehydration, dryness and wrinkle.

[0003] In order to improve the water content of the skin and maintain the elasticity of the skin, moisturizing cosmetics are widely used at present, and moisturizing agents are the key ingredients to achieve the moisturizing effect. Moisturizing agents are based on the simulation of the three components of the skin natural moisturizing system, and can be roughly divided into: (1) oil moisturizing agent: such as vaseline, white wax oil, etc.; (2) hygroscopic moisturizing agent: such as polyhydric alcohol: glycerol, propylene glycol, polyethylene glycol, sorbitol, etc.; (3) hydration moisturizing agent: collagen, hyaluronic acid and other macromolecules from animal bodies; (4) repair moisturizing agent: such as vitamin A, vitamin B, vitamin E and fruit acid, etc. which can remove the outermost layer of the skin that loses the moisturizing function of the stratum corneum, and let the new keratinocytes play the function of the ingredients with repair effect.

[0004] Among them, the macromolecular moisturizing agent such as collagen, hyaluronic acid and other macromolecules from animal bodies has been widely concerned in recent years, in addition, soybean protein, hydrolyzed protein and other protein moisturizing agents have also been reported one after another. But the source of this kind of moisturizing agent is still limited, and it is still of great significance to explore more proteins with moisturizing effect.

[0005] Mussel adhesive protein (MAP) is a protein complex secreted by the foot part of the part of the gland, in recent years, because it has the advantages of high strength, high toughness, degradability, etc. It has been widely concerned, and it also has unique adhesion ability, so it has obtained more and more attention in the field of biological medicine. At present, six kinds of mussel adhesive proteins have been found: foot protein (fp) fp-1, fp-2, fp-3, fp-4, fp-5, fp-6, all of which contain L-3,4-dihydroxyphenylalanine, i.e. DOPA (also derived from tyrosine by hydroxylation), and DOPA is the key factor for the adhesion activity of the protein. However, as a natural source of protein, the uniformity is poor, and the extraction source and actual application are still limited. Therefore, in order to make up for the deficiency of natural mussel adhesive protein, researchers began to study the artificial synthesis of mussel adhesive protein. For example, D.S. Hwang et al. (Biomaterials 28 (2007) 3560-3568) disclosed a mussel adhesive protein fp-151, which has excellent biocompatibility and solubility, and has stronger adhesion capacity than natural mussel adhesive protein.

[0006] However, so far, there have been few reports on the moisturizing effect of natural mussel adhesive protein or artificial mussel adhesive protein. SUMMARY

[0007] The purpose of the present application is to provide a kind of mussel adhesive protein and the use of intermediate as a moisturizing agent with long-acting moisturizing effect.

[0008] The present application provides the use of a protein intermediate with a sequence as shown in SEQ ID NO. 1 in a moisturizing agent.

[0009] The present application also provides the use of a mussel adhesive protein in a moisturizing agent, wherein the mussel adhesive protein is obtained by oxidizing a protein intermediate with a sequence as shown in SEQ ID NO. 1 with tyrosinase.

[0010] Further, the preparation method of the mussel adhesive protein comprises the following steps: adding tyrosinase to a mixed system comprising the protein intermediate of claim 1, a pH adjusting agent and ascorbic acid, reacting, purifying, and obtaining.

[0011] Further, the mixed system further comprises borate; in the mixed system, the concentration of borate is 15-25 mM, the concentration of the pH adjusting agent is 0.05-0.15 mM, and the concentration of ascorbic acid is 5-15 mM; the enzyme activity of tyrosinase is 10-30 U; the reaction time is 4-8 hours, and the temperature is 20-30℃.

[0012] The method for purification is desalination and concentration through an ultrafiltration membrane.

[0013] Further, the borate is sodium borate, the pH regulator is phosphate, the concentration of the borate in the mixed system is 20 mM, the concentration of the pH regulator is 0.1 mM, the concentration of ascorbic acid is 10 mM, the pH of the mixed system is 7.0, the enzyme activity of the tyrosinase is 20 U, the reaction time is 6 hours, the temperature is 25℃, and the molecular weight cut-off of the ultrafiltration membrane is 1000 Da.

[0014] Further, the mussel-like adhesive protein is the mussel-like adhesive protein MLAP316 in the Chinese patent application with the application number CN202210067273.2.

[0015] Further, the humectant is a humectant for daily-use products.

[0016] Further, the daily-use product is a skin care product or a cosmetic product.

[0017] The protein intermediate and the type III mussel-like adhesive protein provided by the application have excellent long-acting moisturizing effect, and have wide application prospects as a humectant.

[0018] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0019] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as the scope of the above subject matter of the application being limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. DETAILED DESCRIPTION

[0020] The glycerol, sodium hyaluronate and collagen used as the comparative examples in the application are known products, which are obtained by purchasing commercially available products.

[0021] Among them, the collagen is type III collagen with a molecular weight of 58 kD, and the sodium hyaluronate is a common daily-use raw material with a molecular weight of 500-730 kD.

[0022] Example 1, preparation of the mussel-like adhesive protein of the application

[0023] The preparation method of the intermediate and the mytilus edulis-like mucin can refer to the preparation method of mlp316 and MLAP316 in the Chinese patent application No. CN202210067273.2 (paragraphs 0029-0052 of the specification). Or prepared by conventional technical means mastered by those skilled in the art, for example, by fermentation of recombinant engineering bacteria or chemical synthesis of protein intermediate, the sequence is (SEQ ID NO. 1):

[0024] MGGNYYPKYKYPRGYKGGYNGYPRGNYGWNKGWKKGRWGRKYYAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKYDGYSDGYYPGSAYNYPSGSHGYHGHGYKGKYYGKGKKYYYKYKRTGKYKYLKKARKYHRKGYKKYYGGGSSAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKGGNYYPKYKYPRGYKGGYNGYPRGNYGWNKGWKKGRWGRKYY.

[0025] Further add the intermediate to the protein intermediate oxidation reaction system containing ascorbic acid, sodium borate and phosphate: the final concentration of ascorbic acid is 10 mM, the final concentration of sodium borate is 20 mM, the final concentration of phosphate is 0.1 mM, adjust the pH to 7.0, add 20 U of tyrosinase, and slowly stir at 25℃ for 6 h. After the reaction is completed, use 0.5% acetic acid solution to top wash the desalted and concentrated in an ultrafiltration membrane with a molecular weight cut-off of 1000 Da. Freeze-dry the concentrated solution to obtain the mytilus edulis-like mucin of type III of the application.

[0026] The beneficial effects of the mytilus edulis-like mucin of the application are demonstrated by the following experimental examples.

[0027] According to the previous experiments of the inventors, the protein intermediate prepared in the application itself has excellent moisturizing performance, and after modification by tyrosinase oxidation, the long-acting moisturizing effect of the obtained mytilus edulis-like mucin is further improved. The following takes the type I mytilus edulis-like mucin prepared in Example 1 as an example to perform verification experiments of the moisturizing performance:

[0028] Experimental Example 1: Determination of Moisturizing Performance of Samples by Gravimetric Method

[0029] Place the test sample and the control sample in a relative humidity of 43% (RH 43%) for 97 h, and calculate the sample moisturizing rate. Among them, the number of sample repeats n = 3, the gravimetric method is used, and the samples are taken at 0 h, 2 h, 4 h, 6 h, 8 h, 24 h, 49 h, 73 h and 97 h, the mass of each sample is accurately weighed, and the moisturizing rate of each sample is calculated by the moisturizing rate calculation formula.

[0030] Moisturizing rate calculation formula:

[0031]

[0032] Wherein:

[0033] W0 is the initial mass of the sample, g;

[0034] W t is the weighed mass of the sample after t h.

[0035] The results of the RH 43% moisturizing experiment are shown in Table 1, and the ranking data of the moisturizing rate of each sample are shown in Table 2.

[0036] Table 1 Moisturizing rate data of five samples after 97 hours (RH = 43%)

[0037]

[0038]

[0039] Table 2 Ranking results of five samples (RH = 43%) after 8, 24, 49, 73 and 97 h

[0040]

[0041] As shown in Table 1, the type III mussel adhesive protein intermediate with the specific sequence of the application has excellent moisturizing effect, and the type III mussel protein after further tyrosinase oxidation modification shows more excellent moisturizing effect. Specifically, the short-term moisturizing performance of each sample is equivalent, and the sodium hyaluronate has the best effect, and the type III mussel adhesive protein is slightly lower than the collagen. The moisturizing rate of the collagen shows a significant downward trend after 49 h, and the type III mussel adhesive protein and the type III mussel adhesive protein intermediate are relatively stable until 97 h. Table 2 is the ranking result of the moisturizing rate of each sample after 8, 24, 49, 73 and 97 h. In the short-term test of 8 h, the sodium hyaluronate has the best effect, and in a slightly longer time, the type III mussel adhesive protein shows a significant advantage, and the type III mussel adhesive protein intermediate is second. From the above test results, it can be seen that the type III mussel adhesive protein and the type III mussel adhesive protein intermediate have good long-term moisturizing effect in vitro.

[0042] Experimental Example 2: Determination of the water content in the stratum corneum of the skin by the capacitance method

[0043] 1. The test sample is 1% sodium hyaluronate + 3% glycerol, 1% type III mussel adhesive protein, 3% glycerol, and deionized water (blank control).

[0044] 2. The subjects are 24 healthy volunteers aged 20-50 years old without skin diseases. During the test period, the temperature and humidity of the environment are required to be constant, the temperature is 20-22℃, and the relative humidity is 30%-60%.

[0045] 3. Test method: The test site is the flexor side of the forearm, and the flexor side of the forearm of both hands is marked with a test area of 3cm*3cm. Three areas are marked on the same arm. Four samples are statistically balanced distributed in each area, and the amount of each area is 0.02mL. The latex finger sleeve is used for uniform coating, and the German CK-skin moisture test probe Corneometer CM825 instrument is used for parallel determination of 5 times. The stratum corneum moisture content is tested before using the sample (0h), 2, 4, 6, 8, 24, 48, 72h after using the sample, and the arithmetic mean value is taken as the test result.

[0046] 4. Statistical method: Using IBM SPSS Statistics 21 software paired t test, if the stratum corneum moisture content of the test area before and after using the product shows significant difference, it indicates that the tested sample has moisturizing effect; otherwise, it indicates that the tested sample does not have moisturizing effect. The results are shown in Tables 4 and 5.

[0047] Table 3 Average moisture content of four samples placed for 72 hours

[0048]

[0049] Table 4 Moisture content change rate (%) of four samples placed for 72 hours

[0050]

[0051] From the data in Tables 3 and 4, it can be seen that after using the three test samples, the skin moisture content increases within 2h, among which the skin moisture content increases most significantly after applying 3% glycerol, and the skin moisture content increases least after applying deionized water. Due to the volatilization of epidermal water, the skin moisture content decreases after 2h. After 24h, the skin moisture content after applying 1% type II mussels mucin increases most significantly compared with before application, which is significantly better than 1% sodium hyaluronate and 3% glycerol. After 72h, the water content of the experimental group using 1% type II mussels mucin is still comparable to that of the control group using 1% sodium hyaluronate.

[0052] From the above test results, it can be seen that type I mussels mucin and its intermediates have excellent long-acting moisturizing effect on human body. As a peptide compound, it has less stimulation than glycerol and other small molecule moisturizing agents. At the same time, compared with natural extracted hyaluronic acid and other natural source macromolecular moisturizing agents, it has better uniformity, which is more conducive to standardized, large-scale and industrialized production. Therefore, the mussels protein and its intermediates as moisturizing agents have broad application prospects.

Claims

1. Use of a Mytilus galloprovincialis mucoprotein for the preparation of an in vitro long-lasting moisturizer, characterized by, The mussel-like adhesive protein is obtained by oxidizing a protein intermediate with a sequence shown in SEQ ID NO. 1 by tyrosinase; The preparation method of the mussel-like adhesive protein comprises the following steps: adding tyrosinase into a mixed system comprising a protein intermediate with a sequence shown in SEQ ID NO. 1, a pH regulator and ascorbic acid, reacting, purifying, and obtaining the mussel-like adhesive protein.

2. Use according to claim 1, characterized in that, The mixed system further comprises borate; in the mixed system, the concentration of the borate is 15-25 mM, the concentration of the pH regulator is 0.05-0.15 mM, and the concentration of the ascorbic acid is 5-15 mM; the enzyme activity of the tyrosinase is 10-30 U; the reaction time is 4-8 hours, and the reaction temperature is 20-30 DEG C. The purifying method is desalination and concentration through an ultrafiltration membrane.

3. Use according to claim 2, characterized in that, The borate is sodium borate, and the pH regulator is phosphate; in the mixed system, the concentration of the borate is 20 mM, the concentration of the pH regulator is 0.1 mM, and the concentration of the ascorbic acid is 10 mM; the pH of the mixed system is 7.0; the enzyme activity of the tyrosinase is 20 U; the reaction time is 6 hours, and the reaction temperature is 25 DEG C; and the molecular weight cut-off of the ultrafiltration membrane is 1000 Da.

4. The use according to claim 1, characterized in that, The moisturizer is a moisturizer used in a daily-use chemical product.

5. Use according to claim 4, characterized in that, The daily-use chemical product is a skin care product or a cosmetic product.

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