A kind of long-acting moisturizing efficacy of mussels like mucin and its intermediate

Abstract

The present application provides a protein intermediate and a kind of mussels' mucin obtained by oxidizing the protein intermediate by tyrosinase.The protein intermediate and the mussels' mucin provided by the present application have excellent long-acting moisturizing effect, and have wide application prospect as moisturizing agent.

Description

Technical Field

[0001] This invention belongs to the field of proteins, specifically relating to a mussel-like adhesive protein with long-lasting moisturizing effects and its intermediates. Background Technology

[0002] Human skin has a natural moisturizing system composed of water, natural moisturizing factor (NMF), and lipids, which plays a vital role in maintaining skin elasticity. NMF's molecular structure is rich in hydroxyl groups, which bind water molecules, while lipids form a thin layer on the skin surface, preventing water molecules from escaping into the environment and preventing moisture evaporation. The content of these three components and their balance have a significant impact on the skin. In autumn and winter, the dry air alters the skin's physiological functions, accelerating moisture evaporation, slowing blood circulation, and reducing sweat excretion and sebum secretion. Therefore, the skin is prone to dehydration, dryness, and wrinkles.

[0003] To improve skin hydration and maintain skin elasticity, moisturizing cosmetics are widely used, and moisturizers are the key ingredients for achieving moisturizing effects. Based on the simulation of three components of the skin's natural moisturizing system, moisturizers can be roughly divided into: (1) oil moisturizers: such as petrolatum, paraffin oil, etc.; (2) humectant moisturizers: such as polyols: glycerin, propylene glycol, polyethylene glycol, sorbitol, etc.; (3) hydrating moisturizers: such as collagen, hyaluronic acid, etc., which are macromolecules derived from animals; (4) repairing moisturizers: such as vitamin A, vitamin B, vitamin E, and fruit acids, which can remove the outermost layer of the skin that has lost its moisturizing function, allowing new keratinocytes to perform their functions and have a repairing effect.

[0004] Among them, large-molecule moisturizers derived from animal bodies, such as collagen and hyaluronic acid, have received widespread attention in recent years. In addition, protein-based moisturizers such as soy protein and hydrolyzed protein have also been reported. However, the sources of these moisturizers are still relatively limited, and exploring more proteins with moisturizing effects remains of great significance.

[0005] Mussel adhesive protein (MAP) is a protein complex secreted by the foot glands of mussels. In recent years, it has attracted widespread attention due to its high strength, high toughness, and biodegradability. Furthermore, its unique adhesive ability has garnered increasing interest in fields such as biomedicine. Currently, six types of mussel adhesive proteins have been identified: foot protein (fp) fp-1, fp-2, fp-3, fp-4, fp-5, and fp-6. All six adhesive proteins contain L-3,4-dihydroxyphenylalanine, i.e., DOPA (derived from tyrosine through hydroxylation), which is a key factor in the adhesive activity of these proteins. However, as naturally occurring proteins, their homogeneity is poor, limiting their extraction sources and practical applications. Therefore, to compensate for the shortcomings of natural mussel adhesive proteins, researchers have begun to study artificially synthesized mussel-like adhesive proteins. For example, DSHwang et al. (Biomaterials 28(2007)3560-3568) disclosed a mussel-like adhesive protein fp-151, which has excellent biocompatibility and solubility, and at the same time has stronger adhesion ability than natural mussel adhesive protein.

[0006] However, to date, there are few reports on the moisturizing effects of either natural mussel adhesive protein or artificially synthesized mussel-like adhesive protein. Summary of the Invention

[0007] The purpose of this invention is to provide a mussel-like adhesive protein and its intermediates with long-lasting moisturizing effects, and its use as a moisturizer.

[0008] This invention provides a protein intermediate, the sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the use of the aforementioned protein intermediates in humectants.

[0010] The present invention also provides a mussel-like adhesive protein, which is obtained by oxidizing the above-mentioned protein intermediate with tyrosinase.

[0011] Furthermore, the preparation method of the above-mentioned mussel-like adhesive protein includes the following steps: adding tyrosinase to a mixed system comprising the protein intermediate described in claim 1, a pH adjuster, and ascorbic acid, reacting, purifying, and thus obtaining the product.

[0012] Furthermore, the above-mentioned mixed system also includes borate; in the mixed system, the concentration of borate is 15-25 mM, the concentration of pH adjuster is 0.05-0.15 mM, 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℃.

[0013] The purification method is: desalting and concentration via ultrafiltration membrane.

[0014] Furthermore, the aforementioned borate is sodium borate, and the pH adjuster is phosphate; in the mixed system, the concentration of borate is 20 mM, the concentration of pH adjuster is 0.1 mM, and the concentration of ascorbic acid is 10 mM; the pH of the mixed system is 7.0; the enzyme activity of tyrosinase is 20 U; the reaction time is 6 hours, and the temperature is 25°C; the molecular weight cutoff of the ultrafiltration membrane is 1000 Da.

[0015] The present invention also provides the use of the above-mentioned mussel-like adhesive protein in humectants.

[0016] The present invention also provides a moisturizer comprising the above-mentioned protein intermediate and / or the above-mentioned mussel-like adhesive protein.

[0017] The present invention also provides the use of the above-mentioned moisturizer in daily chemical products with moisturizing effects.

[0018] Furthermore, the aforementioned daily chemical products are skincare or cosmetic products.

[0019] The beneficial effects of the present invention are as follows: The protein intermediate with a specific amino acid sequence provided by the present invention exhibits excellent moisturizing effect, and the type I mussel adhesive protein obtained by further modification has even better long-lasting moisturizing effect and has broad application prospects as a moisturizer.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0022] The glycerin, sodium hyaluronate, collagen raw materials and equipment used in this invention as comparative examples are all known products, obtained by purchasing commercially available products.

[0023] Among them, the collagen is type III collagen with a molecular weight of 58kD; sodium hyaluronate is a common daily chemical raw material with a molecular weight of 500-730kD.

[0024] Example 1: Preparation of the protein intermediate and mussel-like adhesive protein of the present invention

[0025] The intermediate and mussel-like adhesive protein can be prepared using methods similar to those disclosed in Chinese Patent Application No. CN202210067273.2, paragraphs 0029-0052. Alternatively, they can be prepared using conventional techniques known to those skilled in the art, such as fermentation with recombinant engineered bacteria or chemical synthesis of the protein intermediate, with the sequence (SEQ ID NO.1):

[0026] MAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKAKPSYPPTYK.

[0027] The intermediate was further added to a protein intermediate oxidation reaction system containing ascorbic acid, sodium borate, and phosphate: the final concentration of ascorbic acid was 10 mM, the final concentration of sodium borate was 20 mM, and the final concentration of phosphate was 0.1 mM. The pH was adjusted to 7.0, and 20 U of tyrosinase was added. The reaction was carried out by slow stirring at 25°C for 6 hours. After the reaction was completed, the solution was eluted with 0.5% acetic acid solution through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da and then concentrated. The concentrated solution was lyophilized to obtain the type I mussel adhesive protein of this invention.

[0028] The following experimental examples demonstrate the beneficial effects of the mussel adhesive protein of this invention.

[0029] Based on the inventors' preliminary experiments, the protein intermediate prepared by this invention inherently possesses excellent moisturizing properties. Furthermore, after modification by tyrosinase oxidation, the long-lasting moisturizing effect of the resulting mussel-like adhesive protein is further enhanced. The following is a verification experiment of the moisturizing performance using the type I mussel-like adhesive protein prepared in Example 1 as an example:

[0030] Experimental Example 1: Determination of the moisturizing properties of samples by weighing method

[0031] The test samples and control samples were placed at an ambient temperature of 20℃ and a relative humidity of 43% (RH 43%), and the moisture retention rate of the samples was determined within 96 hours. The number of sample replicates was n=3. Using an analytical balance, samples were taken at 0h, 2h, 4h, 6h, 8h, 20h, 24h, 48h, 72h, and 96h, and the mass of each sample was accurately weighed. The moisture retention rate of each sample was calculated using the moisture retention rate calculation formula.

[0032] Formula for calculating moisturizing rate:

[0033]

[0034] in:

[0035] W0 represents the initial mass of the sample being tested, in grams;

[0036] W t The mass of the sample after th is measured, in g.

[0037] The results of the RH 43% moisturizing experiment are shown in Table 1, and the ranking of the moisturizing rates of each sample is shown in Table 2.

[0038] Table 1. Moisture retention rate data of 5 samples after 96 hours of storage (RH = 43%)

[0039]

[0040]

[0041] Table 2. Ranking of the moisture retention rate (RH = 43%) of the five samples after 8, 24, 48, 72, and 96 hours of storage.

[0042]

[0043] As shown in Table 1, the intermediate of type I mussel adhesive protein with a specific sequence of this invention has excellent moisturizing effect, and the type I mussel adhesive protein after further modification by tyrosinase oxidation exhibits even better moisturizing effect. Specifically, within 24 hours, the moisturizing rate of type I mussel adhesive protein was comparable to that of the control samples; the moisturizing performance of collagen decreased rapidly after 24 hours, while the change trend of type I mussel adhesive protein remained relatively stable until the end of the experiment at 96 hours. Table 2 shows the ranking results of the moisturizing rate of each sample at different test time points. In the short-term test of 8 hours, sodium hyaluronate showed the best effect, while in the slightly longer test time of 72 hours and above, type I mussel adhesive protein had the best moisturizing rate. From the above test results, the moisturizing rate of type I mussel adhesive protein was consistently stable without significant decrease, and after 72 hours, the moisturizing rate of type I mussel adhesive protein remained the highest, indicating that type I mussel adhesive protein has a good long-lasting moisturizing effect in vitro.

[0044] Experimental Example 2: Determination of skin stratum corneum moisture content by capacitance method

[0045] 1. The test sample consisted of 1% sodium hyaluronate, 1% type I mussel adhesive protein (Example 1), and 3% glycerol (deionized water as solvent), and a blank group (deionized water) was set up.

[0046] 2. The subjects were 24 healthy volunteers aged 20-50 years without skin diseases. During the test, the ambient temperature and humidity were kept constant: temperature 20-22℃ and relative humidity 30%-60%.

[0047] 3. Testing Method: The test site was the flexor surface of the forearm. A 3cm x 3cm test area was marked on the flexor surface of both forearms, with three areas marked on each arm. The four types of samples were statistically evenly distributed across the areas. The sample volume for each area was 0.02 mL, applied evenly using a latex finger cot. Each area was measured five times in parallel using a German CK-Corneometer CM825 skin moisture testing probe. The stratum corneum moisture content was measured before sample application (0 h) and at 2, 4, 6, 8, 24, 48, and 72 h after sample application. The arithmetic mean of the results was used.

[0048] 4. Statistical Methods: Paired t-tests were used with IBM SPSS Statistics 21 software. If there is a significant difference in the moisture content of the stratum corneum in the test area before and after product use, it indicates that the test sample has a moisturizing effect; otherwise, if there is no significant difference, it indicates that the test sample does not have a moisturizing effect.

[0049] Table 3. Average moisture content of the four samples after 72 hours of storage.

[0050]

[0051] Table 4. Change rate (%) of moisture content of four samples after 72 hours of storage.

[0052] As shown in Tables 3 and 4, skin moisture content increased within 2 hours after using all three test samples. The increase was most significant with the application of 3% glycerin, while the increase was least significant with the application of deionized water. Due to epidermal water evaporation, skin moisture content decreased after 2 hours in all samples. After 24 hours, the skin treated with 1% type I mussel adhesive protein showed the most significant increase in moisture content compared to before treatment, significantly better than the 1% sodium hyaluronate and 3% glycerin treatments. After 72 hours, the water content of the experimental group using 1% type I mussel adhesive protein remained comparable to that of the control group using 1% sodium hyaluronate.

[0053] The test results above show that type I mussel adhesive protein and its intermediates have excellent long-lasting moisturizing effects on the human body. Furthermore, as a peptide compound, it is less irritating than small-molecule moisturizers such as glycerin. Moreover, compared to naturally derived large-molecule moisturizers such as sodium hyaluronate, it exhibits better uniformity, making it more conducive to standardized, large-scale, and industrialized production. Therefore, the mussel adhesive protein and its intermediates of this invention have broad application prospects as moisturizers.

Claims

1. Use of the protein intermediate with the sequence shown in SEQ ID NO.1 in the preparation of moisturizers.

2. The use of mussel-like adhesive proteins in the preparation of moisturizers, characterized in that, The mussel-like adhesive protein is obtained by oxidizing a protein intermediate with the sequence shown in SEQ ID NO.1 with tyrosinase.

3. The use as described in claim 2, characterized in that, The preparation method of the mussel-like adhesive protein includes the following steps: adding tyrosinase to a mixed system including the protein intermediate, pH adjuster and ascorbic acid, reacting, purifying, and then obtaining the protein.

4. A humectant, characterized in that, It comprises a protein intermediate with the sequence shown in SEQ ID NO.1 and / or a mussel-like adhesive protein, wherein the mussel-like adhesive protein is obtained by oxidizing the protein intermediate with the sequence shown in SEQ ID NO.1 with a tyrosinase.

5. The humectant as described in claim 4, characterized in that: The preparation method of the mussel-like adhesive protein includes the following steps: adding tyrosinase to a mixed system including the protein intermediate, pH adjuster and ascorbic acid, reacting, purifying, and then obtaining the protein.

6. Use of the moisturizer according to claim 4 or 5 in the preparation of daily chemical products with moisturizing effects.

7. The use as described in claim 6, characterized in that, The daily chemical products mentioned are skin care products or cosmetics.

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