Composition with whitening effect, application thereof, and skin care products and cosmetics

Abstract

The present invention discloses a composition with whitening efficacy, comprising a first small molecule hyaluronic acid, wherein the molecular weight of the first small molecule hyaluronic acid is 750-1200Da. The present invention also provides an application of the above-mentioned composition with whitening efficacy in skin care products and skin care products. The present invention also provides an application of the above-mentioned composition with whitening efficacy in cosmetics and cosmetics. The composition with whitening efficacy of the present invention inhibits the activity of tyrosinase and reduces the production of melanin by adopting the first small molecule hyaluronic acid, and has the advantage of significant whitening effect. The composition with whitening efficacy of the present invention has extremely high biocompatibility, does not produce additional irritation to the skin, has a high safety factor, and has a wide range of application scenarios. It can be widely used in the fields of skin care products, cosmetics, etc.

Description

Technical Field

[0001] The present invention belongs to the field of skin care products and cosmetics, and in particular relates to a whitening product and application thereof, as well as skin care products and cosmetics. Background Art

[0002] Fair skin has always been a cherished pursuit of Eastern women, particularly the belief that "whiteness conceals all flaws," which underscores the importance of skin whitening. Whitening involves reducing melanin, evens skin tone, lightens chloasma, and eliminates localized pigmentation. However, the fast pace of modern life can easily lead to skin problems such as dullness and melanin accumulation. For example, lack of sleep in modern life ultimately contributes to melanin accumulation. Generally speaking, skin whitening is primarily related to melanin accumulation.

[0003] Existing whitening products (such as facial masks and skincare products) may contain chemicals that exfoliate the stratum corneum. Improper use can cause thinning of the stratum corneum, resulting in redness, swelling, pain, dryness, and the appearance of red streaks on the skin. Furthermore, use of these whitening products can irritate pores, causing enlarged pores and leading to folliculitis, acne, and other skin conditions. They can also cause edema and allergies. Therefore, the safety of existing whitening products needs to be improved.

[0004] Hyaluronic acid, also known as hyaluronic acid, is a linear high-molecular-weight polysaccharide composed of D-glucuronic acid and N-acetylglucosamine disaccharide units linked alternately by β-1,3- and β-1,4-glycosidic bonds. It has excellent safety. Hyaluronic acid is a major component of the extracellular matrix (ECM) and a key constituent of skin, vitreous humor, synovial fluid, and cartilage. It possesses unique physicochemical properties and biological functions. Research has shown that the biological functions of hyaluronic acid are closely linked to its molecular weight, with hyaluronic acid of different molecular weights exhibiting distinct bioactivities. High-molecular-weight hyaluronic acid exhibits excellent moisturizing, viscoelasticity, lubricity, inflammation suppression, and sustained drug release properties. Low-molecular-weight hyaluronic acid and hyaluronic acid oligosaccharides, on the other hand, promote wound healing, angiogenesis, anti-tumor, and immunomodulatory properties. Hyaluronic acid with very low molecular weight has attracted widespread attention and consumer favor due to its excellent transdermal absorption and biological activity.

[0005] At present, the main functions of hyaluronic acid focus on hydration, anti-oxidation, immune regulation and other functions. There are no clear reports on the research of small molecular weight hyaluronic acid in the whitening dimension. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a composition with whitening efficacy that has a significant whitening effect and a high safety factor, as well as its application, skin care products and cosmetics. To solve the above technical problems, the technical solution proposed by the present invention is as follows:

[0007] A composition with whitening effect comprises a first small molecule hyaluronic acid, wherein the molecular weight of the first small molecule hyaluronic acid is 750-1200Da.

[0008] In the above composition, preferably, the first small molecule hyaluronic acid comprises hyaluronic acid tetrasaccharide and hyaluronic acid hexasaccharide, and the weight ratio of the hyaluronic acid tetrasaccharide to the hyaluronic acid hexasaccharide is (50-80): (20-40).

[0009] In the above composition, preferably, the weight ratio of the hyaluronic acid tetrasaccharide to the hyaluronic acid hexasaccharide is (60-70):(25-30).

[0010] In the above composition, preferably, the composition further comprises a second small molecule hyaluronic acid with a smaller molecular weight, and the molecular weight of the second small molecule hyaluronic acid is 300-450 Da.

[0011] In the above composition, preferably, the second small molecule hyaluronic acid includes hyaluronic acid disaccharide, and the weight ratio of the hyaluronic acid disaccharide to the first small molecule hyaluronic acid is (2-15): (70-120).

[0012] In the above composition, preferably, the weight ratio of the hyaluronic acid disaccharide to the first small molecule hyaluronic acid is (5-10): (85-100).

[0013] As a general technical concept, the present invention also provides an application of the above-mentioned composition with whitening effect in skin care products.

[0014] As a general technical concept, the present invention also provides a skin care product, the components of which include the above-mentioned composition with whitening effect.

[0015] As a general technical concept, the present invention also provides an application of the above-mentioned composition with whitening effect in cosmetics.

[0016] As a general technical concept, the present invention also provides a cosmetic, the components of which include the above-mentioned composition with whitening effect.

[0017] The present invention provides a composition with a significant whitening effect and a high safety factor. Specifically, the present invention starts from the key factors of whitening, tyrosinase activity and melanin production, and explores the whitening efficacy of the hyaluronic acid composition by optimizing the combination of hyaluronic acids of different molecular weights (preferably a combination of a first small molecule hyaluronic acid and a second small molecule hyaluronic acid). The whitening effect of the hyaluronic acid composition is verified by a cell whitening model and a zebrafish whitening model, providing an experimental basis and reference value for the application and efficacy research of the small molecule hyaluronic acid composition.

[0018] Hyaluronic acid disaccharide, tetrasaccharide, hexasaccharide are polysaccharides containing one, two, three hyaluronic acid disaccharide structures (D-glucuronic acid and N-acetylglucosamine) respectively, are typical low-molecular-weight hyaluronic acids. In the prior art, the main effects of hyaluronic acid disaccharide, tetrasaccharide, hexasaccharide focus on the efficacy dimensions such as moisturizing, anti-oxidation. There are no clear and relevant reports for the research on hyaluronic acid disaccharide, tetrasaccharide, hexasaccharide in whitening dimension. In the preferred embodiment of the present invention, the whitening efficacy of hyaluronic acid compositions is explored by optimizing the ratio of hyaluronic acid disaccharide, tetrasaccharide, hexasaccharide. Wherein, hyaluronic acid tetrasaccharide, hexasaccharide have the effect of suppressing tyrosinase activity and melanin production, and hyaluronic acid disaccharide can promote the lifting of this effect, and both cooperate and complement each other, more significantly realizing the suppression of tyrosinase and melanin.

[0019] The specific analysis is as follows:

[0020] Tyrosinase is an oxidase and the rate-limiting enzyme regulating melanin production. Generally, inhibiting tyrosinase activity can effectively limit melanin production, thereby achieving a whitening effect. Therefore, tyrosinase activity and melanin production are key methods for determining whether a raw material or composition has whitening efficacy.

[0021] The present invention studies have shown that hyaluronic acid tetrasaccharide and hexasaccharide refer to small molecule hyaluronic acid composed of two or three disaccharide (D-glucuronic acid and N-acetylglucosamine) units, and its molecular weight is between 750-1200Da. To explore its new efficacy dimension, the present invention carries out tyrosinase inhibition test after configuring hyaluronic acid tetrasaccharide and hexasaccharide powder into aqueous solution. The test results show that the aqueous solution of hyaluronic acid tetrasaccharide and hexasaccharide has significant tyrosinase inhibitory activity, showing its potential in whitening efficacy dimension. To further verify its whitening efficacy, experimental verification was carried out respectively to melanocyte model and zebrafish whitening test model. The result shows that the aqueous solution of hyaluronic acid tetrasaccharide and hexasaccharide can reduce the generation of cell melanin, and can also reduce the generation of melanin in zebrafish, showing significant whitening efficacy. The above results illustrate that hyaluronic acid tetrasaccharide and hexasaccharide have certain application potential in whitening dimension.

[0022] When conducting whitening tests on hyaluronic acid tetrasaccharides and hexasaccharides, hyaluronic acid disaccharides with a molecular weight between 300-450Da were added to the aqueous solution as a whitening enhancer. The test results showed that after the addition of disaccharide, the hyaluronic acid tetrasaccharide and hexasaccharide composition had a higher tyrosinase inhibition rate and melanin inhibition rate, demonstrating its role as a whitening enhancer.

[0023] The composition with whitening efficacy of the present invention focuses on the activity of tyrosinase and the generation of melanin, and suppresses the activity of tyrosinase and reduces the generation of melanin by selecting hyaluronic acid tetrasaccharide and hexasaccharide, and further suppresses the activity of tyrosinase and reduces melanin by adding hyaluronic acid disaccharide, thereby achieving skin whitening from the source. At the same time, it can avoid the stimulation of the body by the whitening ingredients of benzene ring and its derivatives, achieve the purpose of gentle whitening, have the advantages of significant whitening effect, gentle and non-irritating, and wide range of application, and can be widely used in whitening skin care products and whitening cosmetics. In addition, hyaluronic acid disaccharide, tetrasaccharide and hexasaccharide have extremely high biocompatibility, will not produce additional stimulation to the skin, and have extremely high safety.

[0024] The raw materials mentioned in the present invention can be conventional raw materials in the prior art, such as hyaluronic acid disaccharide, tetrasaccharide, and hexasaccharide can all be enzyme-cut hyaluronic acid produced by our company. For details, please refer to patent application CN114574532A.

[0025] The other components in the whitening skin care product and whitening cosmetics of the present invention are not limited and conventional components in the prior art may be used.

[0026] Compared with the prior art, the advantages of the present invention are:

[0027] 1. The composition with whitening efficacy of the present invention inhibits the activity of tyrosinase and reduces the production of melanin by using the first small molecule hyaluronic acid, and has the advantage of a significant whitening effect.

[0028] 2. The compositions with whitening effects of the present invention have extremely high biocompatibility, will not cause additional irritation to the skin, have a high safety factor, and have a wide range of application scenarios. They can be widely used in skin care products, cosmetics and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1The figure shows the results of tyrosinase inhibition rate of B16 cells in Example 1-7 and Comparative Example 1-5.

[0031] Figure 2 This is a graph showing the inhibition rate of melanin production in B16 cells in Examples 1-7 and Comparative Examples 1-5.

[0032] Figure 3 This is a graph showing the inhibition rate of melanin production in zebrafish embryos in Example 1-7 and Comparative Example 1-5. DETAILED DESCRIPTION

[0033] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0034] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0036] Example 1:

[0037] A composition having whitening efficacy comprises a first small molecule hyaluronic acid and a second small molecule hyaluronic acid, wherein the first small molecule hyaluronic acid comprises hyaluronic acid tetrasaccharide and hyaluronic acid hexasaccharide, and the second small molecule hyaluronic acid comprises hyaluronic acid disaccharide, with the weight proportions of hyaluronic acid disaccharide, hyaluronic acid tetrasaccharide, and hyaluronic acid hexasaccharide being 4%, 64%, and 32%, respectively. The details are shown in Table 1 below.

[0038] Table 1: Raw material ratios (mass ratios) of the compositions with whitening efficacy in Examples 1-7 and Comparative Examples 1-5

[0039]

[0040] The preparation of hyaluronic acid disaccharides, tetrasaccharides, and hexasaccharides can be referred to patent application CN114574532A. Accurately weigh the hyaluronic acid disaccharides, tetrasaccharides, and hexasaccharides, dissolve them in deionized water, stir them evenly, and filter them through a 0.22 μm inorganic membrane to obtain the hyaluronic acid composition. Hyaluronic acid pentasaccharides and octasaccharides are conventional commercial products.

[0041] Example 2-7:

[0042] The whitening compositions in Examples 2-7 differ from those in Example 1 primarily in the ratio of raw materials, as shown in Table 1 above.

[0043] Comparative Examples 1-5:

[0044] The whitening compositions in Comparative Examples 1-5 differ from those in Example 1 mainly in the different ratios of raw materials, as shown in Table 1 above.

[0045] The effects of the compositions with whitening efficacy in Examples 1-7 and Comparative Examples 1-5 were verified as follows:

[0046] ① Inhibition test of B16 cell tyrosinase by the composition with whitening effect

[0047] 1) Test background:

[0048] Ultraviolet radiation activates tyrosinase in melanocytes. As melanin production increases, skin tone darkens. Therefore, tyrosinase is key to controlling melanocyte activity and determining the rate of melanin synthesis. Screening for tyrosinase inhibitors could lead to the development of skin-whitening products.

[0049] 2) Test method:

[0050] A. Cell inoculation: Adjust the density of B16 cell suspension to 5×10 4 After the cells / mL were inoculated into 6-well plates, 2 mL / well, the plates were placed in a 37°C, 5% CO2 incubator for 24 h.

[0051] B. Dilution and Addition of Test Samples: Prepare the test sample using complete medium as the diluent. Add the whitening composition from the comparative example and the example to each 100 mL of complete medium (see Table 1 for the amount added, in mg; e.g., when adding the composition from Example 1, 20 mg was added to 100 mL of complete medium) to obtain the test sample. Remove the cell fluid from the well plate and add the test sample at a rate of 2 mL / well to form the sample group. Add complete medium to form the negative group. Incubate in a 37°C, 5% CO2 incubator for 48 h.

[0052] C. Intracellular tyrosinase activity assay: Discard the cell supernatant and rinse once with PBS. Resuspend the cells in 200 μL of PBS buffer containing 1% Triton X-100. Place in a -80°C freezer for 1 hour, then thaw at room temperature to lyse the cells. Pipet to dislodge the cells. Directly add 200 μL of 0.1% L-DOPA and mix thoroughly by pipetting. Pipette 100 μL / well into a 96-well plate, plate in triplicate, and incubate at 37°C overnight. Measure the absorbance of each well at 475 nm.

[0053] 3) Test results:

[0054] The calculation formula of B16 cell tyrosinase inhibition rate is: T% = (T c -T x ) / T c *100%;

[0055] Among them, T represents the B16 cell tyrosinase inhibition rate, T x Indicates the OD value of the sample group after B16 cell test, T c It represents the OD value of the negative group after B16 cell test.

[0056] The data are sorted and the following table 2 and Figure 1 .

[0057] Table 2: Tyrosinase inhibition rate of B16 cells

[0058]

[0059]

[0060] from Figure 1 It can be seen from Table 2 that the hyaluronic acid composition provided by the present invention inhibits the activity of B16 cell tyrosinase to a certain extent, and the addition of hyaluronic acid disaccharide effectively improves the inhibition rate of B16 cell tyrosinase. By comparing the B16 cell tyrosinase inhibition rate between each comparative example and the embodiment, it can be seen that there is a synergistic effect between the components of the composition of the present invention. When some components are missing, the efficacy of the composition is significantly reduced.

[0061] ② Test on the inhibition of melanin in B16 cells by the composition with whitening effect

[0062] 1) Test background:

[0063] Melanin is a high-molecular-weight biological pigment primarily synthesized by melanocytes located in the basal layer of the epidermis. It is distributed in the dermis and determines skin color. The distribution of melanin in the dermis determines skin color, and excessive pigmentation is the primary cause of skin spots.

[0064] 2) Test method:

[0065] A. Cell inoculation: Adjust the density of B16 cell suspension to 5×10 4 After the cells / mL were inoculated into 6-well plates, 2 mL / well, the plates were placed in a 37°C, 5% CO2 incubator for 24 h.

[0066] B. Dilution and Addition of Test Samples: Prepare the test sample using complete medium as the diluent. Add the whitening composition from the comparative example and the example to each 100 mL of complete medium (see Table 1 for the amount added, in mg; e.g., when adding the composition from Example 1, 20 mg was added to 100 mL of complete medium) to obtain the test sample. Remove the cell fluid from the well plate and add the test sample at a rate of 2 mL / well to form the sample group. Add complete medium to form the negative group. Incubate in a 37°C, 5% CO2 incubator for 48 h.

[0067] C. Determination of intracellular melanin content: Discard the supernatant and rinse once with PBS. Harvest the cells with trypsin and centrifuge to remove the supernatant. Resuspend the cell pellet in 400 μL of 1 M NaOH (10% DMSO). Incubate in an 80°C water bath for 3.5 h. Transfer the supernatant to a 96-well plate and measure the absorbance of each well at 405 nm.

[0068] 3) Test results

[0069] The calculation formula of B16 cell melanin production inhibition rate is: M% = (M c -M x ) / M c *100%;

[0070] Among them, M represents the inhibition rate of melanin production in 16 cells, M x Indicates the OD value of the sample group after B16 cell test, T c It represents the OD value of the negative group after B16 cell test.

[0071] The data are sorted and the following table 3 and Figure 2 .

[0072] Table 3: B16 cell melanin production inhibition rate

[0073]

[0074] from Figure 2 As can be seen from Table 3, the hyaluronic acid composition provided by the present invention inhibits the production of melanin in B16 cells to a certain extent, and the addition of the enhancer effectively inhibits the production of melanin. By comparing the melanin production inhibition rates between the comparative examples and the examples, it can be seen that the components of the composition of the present invention have a synergistic effect. When some components are missing, the efficacy of the composition is significantly reduced.

[0075] ③ Experiment on the inhibition of melanin in zebrafish embryos by a composition with whitening effect

[0076] 1) Test background:

[0077] Melanin is a high-molecular-weight biological pigment primarily synthesized by melanocytes located in the basal layer of the epidermis. It is distributed in the dermis and determines skin color. The distribution of melanin in the dermis determines skin color, and excessive pigmentation is the primary cause of skin spots.

[0078] 2) Experimental principle:

[0079] During development, zebrafish embryos accumulate melanin, forming their distinctive body color. Under blank or solvent control conditions, 6-8 hpf zebrafish embryos were placed in samples of varying concentrations and incubated at 28°C + 0.5°C for 48 hours. The efficacy of the samples was evaluated by observing changes in the distribution of melanin granules and the deposition of melanin on the embryonic surface.

[0080] 3) Test method:

[0081] A. Procurement of Experimental Animals: Zebrafish were maintained in a water temperature of 25±1°C. On the evening before spawning, females were selected and placed in a breeding box with healthy, large males at a ratio of 1:2. The females were separated from the males by a partition. The partition was opened the next day after the start of the light cycle. After in vitro fertilization between the females and males, the zebrafish embryos were collected. Successfully fertilized embryos were washed twice with culture water and placed in a 90 mm Petri dish filled with Holt-Buffer medium and cultured at 28.5±0.5°C.

[0082] B. Sample Preparation and Dilution: The test sample was prepared using Holt-Buffer culture medium as the diluent. The composition with whitening efficacy in the comparative example and the embodiment was added to the Holt-Buffer culture medium in the following amounts: the composition in each embodiment and the comparative example was added to each 100 mL of Holt-Buffer culture medium (see Table 1 for the amount added, the unit of mass is mg; for example, when adding the composition in Example 1, 20 mg was added to 100 mL of Holt-Buffer culture medium). The test sample was obtained and used to carry out the test, which was the sample group. The test using Holt-Buffer culture medium was the negative group.

[0083] C. Experimental Procedure: Healthy, uniformly developed zebrafish embryos (6-8 hpf) were randomly selected and placed in 96-well plates, with 5 embryos per well. 200 μL of the test sample or Holt-Buffer were added to each well, with triplicate wells for each concentration. The cells were incubated at 28.5 ± 0.5°C. After 48 hours of incubation, the embryos were observed under a microscope for pigmentation and photographed. Images were analyzed for grayscale value using Image J software, and relative melanin content was calculated.

[0084] D. Data processing: According to the calculation formula: relative melanin content = melanin content of control group / melanin content of experimental group × 100%, where the melanin content is the total area containing melanin in the gray value analysis.

[0085] 4) Test results:

[0086] The formula for calculating the inhibition rate of melanin production in zebrafish embryos is: Z% = (Z c -Z x ) / Z c *100%;

[0087] Among them, Z represents the inhibition rate of melanin production in zebrafish embryos, Z x Z represents the relative content of melanin in the sample group after the zebrafish experiment. c It represents the relative content of melanin in the negative group after zebrafish test.

[0088] The data are sorted and the following table 4 and Figure 3 .

[0089] Table 4: Inhibition rate of melanin production in zebrafish embryos

[0090]

[0091] from Figure 3 As can be seen from Table 4, the hyaluronic acid composition provided by the present invention inhibits the production of zebrafish embryo melanin to a certain extent, and the addition of the enhancer effectively inhibits the production of zebrafish melanin. By comparing the zebrafish melanin inhibition rates between the comparative examples and the examples, it can be seen that the components of the composition of the present invention have a synergistic effect. When some components are missing, the efficacy of the composition is significantly reduced.

[0092] The above test results show that the whitening compositions in Examples 1-7 have significant whitening effects and can be widely used in skin care products and cosmetics. When used, they can be added to skin care products and cosmetics in the form of aqueous solutions. The other components of skin care products and cosmetics can be existing conventional components and are not limited in the present invention.

Claims

1. A composition with whitening effect, characterized in that, Comprising a first small molecule hyaluronic acid, wherein the molecular weight of the first small molecule hyaluronic acid is 750-1200 Da; The first small molecule hyaluronic acid comprises hyaluronic acid tetrasaccharide and hyaluronic acid hexasaccharide, and the weight ratio of the hyaluronic acid tetrasaccharide to the hyaluronic acid hexasaccharide is (50-80): (20-40); The composition further comprises a second small molecule hyaluronic acid with a smaller molecular weight, wherein the second small molecule hyaluronic acid has a molecular weight of 300-450 Da; The second small molecule hyaluronic acid includes hyaluronic acid disaccharide, and the weight ratio of the hyaluronic acid disaccharide to the first small molecule hyaluronic acid is (2-15): (70-120).

2. The composition according to claim 1, characterized in that The weight ratio of the hyaluronic acid tetrasaccharide to the hyaluronic acid hexasaccharide is (60-70): (25-30).

3. The composition according to claim 1, characterized in that The weight ratio of the hyaluronic acid disaccharide to the first small molecule hyaluronic acid is (5-10): (85-100).

4. Use of the composition with whitening effect according to any one of claims 1 to 3 in skin care products.

5. A skin care product, characterized in that: The components thereof include the composition with whitening efficacy described in any one of claims 1 to 3.

6. Use of the composition with whitening effect according to any one of claims 1 to 3 in cosmetics.

7. A cosmetic, characterized in that: The components thereof include the composition with whitening efficacy described in any one of claims 1 to 3.

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