A skin-whitening composition that reduces aging melanocytes and its application

Abstract

This invention discloses a skin-whitening composition that reduces aging melanocytes and its application, relating to the cosmetics field. The invention provides a composition that, by reducing aging melanocytes and decreasing SASP secretion, reduces melanin production and inhibits tyrosinase activity while maintaining healthy melanocytes, thus maintaining skin homeostasis while whitening the skin. This composition can be widely used in various skincare products to effectively achieve long-lasting whitening effects.

Description

Technical Field

[0001] This invention relates to the field of cosmetics, and more specifically, to a whitening composition that reduces aging melanocytes and its application. Background Technology

[0002] With social development and improved living standards, people's demand for skin health and beauty is increasing. Skin whitening, as an important part of beauty, is receiving more and more attention.

[0003] While most existing skin-whitening products can improve skin tone to some extent, they have the following limitations: 1. Short-lasting effects: The effects of many skin-whitening products gradually disappear after discontinuation of use. 2. Skin irritation: Some ingredients may cause skin irritation or allergic reactions. 3. Uneven whitening: It is difficult to achieve an even distribution of skin pigment, easily leading to uneven pigmentation. 4. No anti-aging effect: Some products only inhibit tyrosinase activity and do not have an anti-aging effect on melanocytes.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a skin-whitening composition that reduces aging melanocytes and its application.

[0006] This invention is implemented as follows:

[0007] In a first aspect, embodiments of the present invention provide a composition whose active ingredients include: sodium mannose phosphate, nicotinamide, and hydrolyzed conchiolin.

[0008] Secondly, embodiments of the present invention provide a cosmetic product whose active ingredients include the composition described in the foregoing embodiments.

[0009] Thirdly, embodiments of the present invention provide a cosmetic product whose active ingredients include the composition described in the foregoing embodiments.

[0010] Fourthly, embodiments of the present invention provide the application of the compositions described in the foregoing embodiments in the preparation of whitening products.

[0011] The present invention has the following beneficial effects:

[0012] 1. Inhibit melanin production at its source: This composition reduces aging melanocytes and maintains cell homeostasis, thereby reducing melanin production at the cellular level and achieving a lasting whitening effect from the root.

[0013] 2. Multiple application forms: This composition can be widely used in various skin care products, such as face cream, serum, lotion, and mask, making it convenient for consumers to choose suitable products according to different needs;

[0014] 3. Significant whitening effect: This composition reduces aging melanocytes and SASP secretion, maintaining healthy skin. It can effectively reduce skin problems such as age spots and dullness, significantly improve uneven skin tone, and make the skin brighter and more translucent.

[0015] 4. Improve skin health: This composition maintains skin health homeostasis, improves the overall condition of the skin, enhances the skin barrier function, and resists the damage of harmful external substances through the synergistic effect of multiple active ingredients. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 For cytotoxicity experiments;

[0018] Figure 2 For β-galactosidase staining experiment;

[0019] Figure 3 The figure represents the amount of melanin produced, with the model group stimulated with supernatant of senescent melanocytes.

[0020] Figure 4 The inhibition rate of tyrosinase activity of compositions 1-9;

[0021] Figure 5 The melanin production inhibition rate of compositions 1-9;

[0022] Figure 6 The tyrosinase activity inhibition rate of composition 10-15; wherein, composition 10-15 corresponds sequentially to the compositions provided in proportions 1-6;

[0023] Figure 7 The melanin production inhibition rate of composition 10-15; wherein, composition 10-15 corresponds sequentially to the compositions provided in proportions 1-6. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] Skin color is primarily determined by the production and distribution of melanin, which is produced by melanocytes in the skin. Melanocyte aging and dysfunction are significant factors leading to dull skin and age spots. As we age, melanocytes experience functional decline, manifested as reduced or uneven melanin synthesis, resulting in age spots, dullness, and other signs of aging. The main causes of melanocyte aging include: 1. Oxidative stress: The accumulation of free radicals and reactive oxygen species (ROS) damages melanocyte DNA and organelles, leading to decreased function. 2. Ultraviolet radiation damage: UV radiation induces excessive activation of melanocytes; prolonged exposure can cause cell aging and death. 3. Inflammatory response: Chronic inflammation can alter the melanocyte environment, promoting aging. 4. Genetic factors: Mutations or abnormal expression of certain genes can accelerate the aging process of melanocytes.

[0026] Current skin whitening methods mainly focus on inhibiting melanin production, accelerating melanin metabolism, and removing melanin from the skin's surface, such as using tyrosinase inhibitors, antioxidants, fruit acids, and vitamin C. While these methods can improve skin tone to some extent, they have the following limitations: short-lasting effects, skin irritation, and uneven whitening.

[0027] This invention provides a novel composition that can reduce aging melanocytes, thereby reducing SASP secretion, inhibiting tyrosinase activity, and reducing melanin production, fundamentally solving the problem of pigmentation and achieving a whitening effect.

[0028] On one hand, embodiments of the present invention provide a composition whose active ingredients include: sodium mannose phosphate, nicotinamide, and hydrolyzed conchiolin.

[0029] In some embodiments, the active ingredients of the composition, by weight, include: 0.1 to 2 parts sodium mannose phosphate, 20 to 100 parts nicotinamide, and 0.4 to 5 parts hydrolyzed conchiolin.

[0030] Optionally, in the composition, the weight parts of sodium mannose phosphate can be any one or any two of the following: 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, and 2 parts.

[0031] Optionally, the weight parts of nicotinamide in the composition can be any one or any two of the following: 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and 100 parts.

[0032] Hydrolyzed conchiolin is a compound extracted and isolated from the shells of aquatic organisms. The composition provided in the embodiments of the present invention can be selected from hydrolyzed conchiolin extracted by existing publicly available processes or commercially available hydrolyzed conchiolin.

[0033] In some embodiments, the protein content of the hydrolyzed conchiolin is 85 wt% to 100 wt%. The pH of the aqueous solution of the hydrolyzed conchiolin at 25°C is 4.5 to 7.5. As an example, the protein content of the hydrolyzed conchiolin is any one or any two of the following: 85 wt%, 86 wt%, 87 wt%, 88 wt%, 89 wt%, 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, 97 wt%, 98 wt%, 99 wt%, and 100 wt%. As an example, the pH of a 10 wt% aqueous solution of hydrolyzed conchiolin at 25°C can be any one or any two of the following: 4.5, 5, 5.5, 6, 6.5, 7, and 7.5.

[0034] Optionally, the weight parts of hydrolyzed conchiolin in the composition can be any one or any two of the following: 0.4, 0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, and 5 parts.

[0035] In some embodiments, the active ingredients of the composition, by weight, include: 0.1 to 2 parts sodium mannose phosphate, 20 to 100 parts nicotinamide, and 0.4 to 2 parts hydrolyzed conchiolin.

[0036] In some embodiments, the active ingredients of the composition, by weight, include: 0.1 to 1 part sodium mannose phosphate, 40 to 100 parts nicotinamide and 0.4 to 1 part hydrolyzed conchiolin.

[0037] On the other hand, embodiments of the present invention provide a cosmetic product whose active ingredients include the composition described in any of the foregoing embodiments.

[0038] On the other hand, embodiments of the present invention provide a skin care product whose active ingredients include the composition described in any of the foregoing embodiments.

[0039] On the other hand, embodiments of the present invention provide the use of the composition as described in any of the foregoing embodiments in at least one of the following (1) to (3);

[0040] (1) To reduce senescent melanocytes or inhibit the increase of senescent melanocytes for non-disease treatment purposes;

[0041] (2) Maintaining the homeostasis of healthy melanocytes for non-disease treatment purposes;

[0042] (3) Reducing melanin production for non-disease treatment purposes.

[0043] Furthermore, embodiments of the present invention also provide the use of the compositions described in any of the foregoing embodiments in whitening for non-disease treatment purposes or in the preparation of whitening products.

[0044] In some embodiments, the product includes cosmetics or skincare products.

[0045] In some embodiments, the skin care product includes any one of the following: lotion, face cream, eye cream, face mask, hydrating spray, body lotion, and hand cream.

[0046] In some embodiments, the cosmetic product includes either a toner or a foundation.

[0047] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0048] Examples 1-9

[0049] Examples 1 to 9 provide nine sets of compositions. The compositions have the same components, but differ in the ratio (weight ratio) between the components, as shown in Table 1.

[0050] Table 1 Composition of the composition

[0051]

[0052]

[0053] Note: The hydrolyzed conchiolin protein (INCI) contains 100 wt%, and its aqueous solution has a pH of 7.4 at 25°C. The preparation method is described in Example 2 of the patent application document CN111850073A entitled "Pearl Oyster Whitening Factor, Its Preparation Method and Use".

[0054] Comparative Examples 1-6

[0055] Comparative Examples 1-6 provide compositions that differ in that the components and / or the proportions (weight ratios) between the components are different, as shown in Table 2.

[0056] Table 2 Composition of the Composition

[0057]

[0058] Experimental reagents and consumables

[0059]

[0060]

[0061] Experimental instruments

[0062] Equipment Name brand model Biosafety cabinet Thermo Fisher Scientific <![CDATA[MSC-Advantage TM ]]> Cell incubator Thermo Fisher Scientific BB150 centrifuge Thermo Fisher Scientific Sorvall ST8 Refrigerated centrifuge Thermo Fisher Scientific H1650 Inverted microscope Olympus CKX41 Cell counter Countstar Mira FL Ultraviolet irradiator VILBER BIO-SUN ELISA reader BioTek Synergy LX

[0063] Experimental Example 1

[0064] The effects of the compositions of Examples 1-9 on the cytotoxicity of normal human melanocytes were tested.

[0065] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 30 seconds. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0066] 2. Cell seeding: Adjust the cell density to 5×10⁶ cells / year. 4 Cells / mL were seeded into 96-well plates using a multi-pipe technique, 100 μL per well. An equal volume of cell-free culture medium was added to the control group, and an equal volume of PBS was added to the outermost well to prevent evaporation of the culture medium from affecting the experimental results. Finally, the plates were incubated in an incubator for 24 h.

[0067] 3. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0068] 4. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 1.

[0069] 5. Sample loading: Drug administration was initiated when the cell seeding rate in the 96-well plate reached 40%–60%. For the solvent control group, 100 μL of 254 culture medium was added to each well; for the sample group, 100 μL of culture medium containing the corresponding sample concentration was added to each well; for the blank group, no cells were seeded, only 100 μL of cell culture medium was added. After treatment, the 96-well plate was placed in an incubator (37℃, 5% CO2) and incubated for 24 h.

[0070] 6. Detection: Dilute the CCK8 reagent with complete culture medium at a ratio of 1:10 and mix thoroughly. Remove the old culture medium from the 96-well cell culture plate, add 100 μL of the diluted CCK8 reagent to each well, and incubate for 2 hours. Turn on the microplate reader, set the wavelengths to dual wavelengths (Lm1 at 450 nm and Lm2 at 650 nm), and perform the detection.

[0071] 7. Data processing: Calculate the average absorbance values ​​of the blank group and the control group, and record them as Ab and Ac, respectively. The formula for calculating cell viability is: Cell viability (%) = (Absorbance value - Ab) / (Ac - Ab) × 100%.

[0072] See results Figure 1 The composition provided in this embodiment of the invention is non-toxic to human primary melanocytes.

[0073] Experimental Example 2

[0074] The effects of the compositions of Examples 1-9 on the aging of normal human melanocytes were tested.

[0075] 1. Cell Seeding: Remove cells from the incubator, discard the old culture medium, and wash once with 10 mL of PBS. After discarding the PBS, add 1 mL of trypsin and incubate for approximately 30 seconds. After digestion, add 3 mL of culture medium to stop the digestion. Collect the cells into a 15 mL centrifuge tube using a pipette, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the cells in 1 mL of culture medium. After thorough mixing, mix 10 μL of the cell suspension with 10 μL of trypan blue and count the cells using a cell counter. Take a new centrifuge tube, add fresh culture medium according to the required volume, then add the cell suspension to adjust the cell density to 2.5 × 10⁻⁶ cells / mL. 4 Inoculate per mL. After thorough mixing, inoculate into 24-well plates and incubate for 24 hours.

[0076] 2. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0077] 3. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 1.

[0078] 4. Sample addition treatment: When the cell deposition rate in the 24-well plate reaches 30%–40%, administer the drug. Add 500 μL of 254 culture medium to each well of the solvent control group and the model group; add 500 μL of culture medium containing the corresponding concentration of sample to each well of the sample group; after treatment, place the 24-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0079] 5. Modeling treatment: Remove cells from the incubator, discard the culture medium, and wash once with 1 mL of PBS. After discarding the PBS, add 300 μL of PBS and wash with UVB (30 mJ / cm²). 2 Irradiate the sample, remove the PBS, add 500 μl of culture medium, and continue culturing for 24 h.

[0080] 6. β-Galactosidase Staining: Discard the old culture medium, wash once with PBS, add 300 μL of β-Gal fixative, and fix at room temperature for 15 min. Aspirate the cell fixative, wash the cells three times with PBS for 3 min each time, and prepare the staining working solution according to the ratio (B:C:D:E = 5:1:1:93). Aspirate the β-Gal washing solution, add 300 μL of staining working solution to each tube, incubate at 37°C overnight, remove the staining working solution, add 1 ml of PBS, and observe the staining under a microscope.

[0081] 7. Data processing: Count the number of stained cells under the microscope and the total number of cells. SA-β-Gal staining (%) = number of stained cells / total number of cells * 100%.

[0082] See results Figure 2 The composition provided in this embodiment of the invention significantly reduces the staining of β-galactosidase after UVB irradiation, indicating that the composition has the effect of reducing senescent melanocytes.

[0083] Experimental Example 3

[0084] To test the effect of supernatant from senescent melanocytes on melanin production in mouse melanoma cells.

[0085] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 1 min. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0086] 2. Cell seeding: Adjust the cell density to 11 × 10⁶ cells / year. 4 After thoroughly mixing, inoculate 2 mL of the solution into each well of a 6-well plate. Finally, incubate in an incubator for 24 hours.

[0087] 3. Sample processing: Centrifuge the supernatant of normal melanocytes and the supernatant of senescent melanocytes at 1000 rpm for 20 min, collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, add 200 μl to each well, and incubate in an incubator for 24 h.

[0088] 4. Melanin Detection: Discard the supernatant, wash once with PBS, and add 200 μL of trypsin to each well for approximately 1 min. After digestion, add 1 mL of culture medium to stop the digestion, collect the cells into 1.5 mL centrifuge tubes, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, and wash twice more with PBS. After discarding the supernatant, add 120 μL of 10% DMSO solution (prepared with 1M NaOH), vortex thoroughly, and place in an 80℃ metal bath for 30 min. Mix well by pipetting, and then transfer 100 μL of the lysis buffer to a 96-well plate. Measure the absorbance at 405 nm using a microplate reader.

[0089] See results Figure 3 The group that added supernatant of senescent melanocytes produced 23.9% more melanin than the normal group, proving that senescent melanocytes cause surrounding normal melanocytes to produce a large amount of melanin.

[0090] Test Example 4

[0091] The effects of the compositions of Examples 1-9 on the activity of melanocyte tyrosinase were tested.

[0092] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 1 min. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0093] 2. Cell seeding: Adjust the cell density to 11 × 10⁶ cells / year. 4 Cells / mL were seeded into 96-well plates using a multi-pipe technique, 100 μL per well. An equal volume of cell-free culture medium was added to the control group, and an equal volume of PBS was added to the outermost well to prevent evaporation of the culture medium from affecting the experimental results. Finally, the plates were incubated in an incubator for 24 h.

[0094] 3. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0095] 4. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 1.

[0096] 5. Sample loading: When the cell seeding rate in the 96-well plate reaches 40%–60%, administer the drug. For the solvent control group, add 100 μL of culture medium per well; for the sample group, add 100 μL of culture medium containing the corresponding concentration of the sample per well; for the blank group, no cells are seeded, only 100 μL of cell culture medium is added. After treatment, incubate the 96-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0097] 6. Detection: Dilute Triton X-100 to a 1% concentration with sterile water. Discard the old culture medium using a multipipeline and wash twice with PBS. Add 80 μL of 1% Triton X-100 solution to each well and immediately freeze at -80°C for 30 min. Then, thaw at room temperature to allow complete cell lysis. After preheating at 37°C for 5 min, add 20 μL of 1% L-DOPA solution to each well and incubate at 37°C for 2 h. Finally, measure the absorbance at 490 nm using a microplate reader.

[0098] 7. Data Processing: The formula for calculating the tyrosinase activity inhibition rate is: Y = (1 - (As - Ab) / (Ac - Ab)) * 100% (Y: tyrosinase activity inhibition rate; As: absorbance of the sample well; Ab: average absorbance of the blank group; Ac: average absorbance of the control group).

[0099] See results Figure 4 The addition of the composition significantly reduced the activity of tyrosinase in mouse melanoma cells, indicating that the composition has an inhibitory effect on tyrosinase activity.

[0100] Experimental Example 5

[0101] The effects of the compositions of Examples 1-9 on melanin production in mouse melanoma cells were tested.

[0102] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 1 min. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0103] 2. Cell seeding: Adjust the cell density to 11 × 10⁶ cells / year. 4 After thoroughly mixing, inoculate 2 mL of the solution into each well of a 6-well plate. Finally, incubate in an incubator for 24 hours.

[0104] 3. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0105] 4. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 1.

[0106] 8. Sample addition: When the cell deposition rate in the 6-well plate reaches 40%–60%, administer the drug. Add 2 mL of culture medium containing the corresponding concentration of the sample to each well of the sample group; replace the blank group with fresh complete culture medium. After treatment, place the 6-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0107] 5. Melanin Detection: Discard the supernatant, wash once with PBS, and add 200 μL of trypsin to each well for approximately 1 min. After digestion, add 1 mL of culture medium to stop the digestion, collect the cells into 1.5 mL centrifuge tubes, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, and wash twice more with PBS. After discarding the supernatant, add 120 μL of 10% DMSO solution (prepared with 1M NaOH), vortex thoroughly, and place in an 80℃ metal bath for 30 min. Mix well by pipetting, and then transfer 100 μL of the lysis buffer to a 96-well plate. Measure the absorbance at 405 nm using a microplate reader.

[0108] See results Figure 5 The inhibition rate of melanin production produced by adding compositions 1 to 9 was increased compared with that of the blank group, indicating that the compositions have the effect of inhibiting melanin production.

[0109] Experimental Example 6

[0110] The effects of the compositions in Comparative Examples 1–6 on the activity of melanocyte tyrosinase were tested.

[0111] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 1 min. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0112] 2. Cell seeding: Adjust the cell density to 11 × 10⁶ cells / year. 4 Cells / mL were seeded into 96-well plates using a multi-pipe technique, 100 μL per well. An equal volume of cell-free culture medium was added to the control group, and an equal volume of PBS was added to the outermost well to prevent evaporation of the culture medium from affecting the experimental results. Finally, the plates were incubated in an incubator for 24 h.

[0113] 3. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0114] 4. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 2.

[0115] 5. Sample loading: When the cell seeding rate in the 96-well plate reaches 40%–60%, administer the drug. For the solvent control group, add 100 μL of culture medium per well; for the sample group, add 100 μL of culture medium containing the corresponding concentration of the sample per well; for the blank group, no cells are seeded, only 100 μL of cell culture medium is added. After treatment, incubate the 96-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0116] 6. Detection: Dilute Triton X-100 to a 1% concentration with sterile water. Discard the old culture medium using a multipipeline and wash twice with PBS. Add 80 μL of 1% Triton X-100 solution to each well and immediately freeze at -80°C for 30 min. Then, thaw at room temperature to allow complete cell lysis. After preheating at 37°C for 5 min, add 20 μL of 1% L-DOPA solution to each well and incubate at 37°C for 2 h. Finally, measure the absorbance at 490 nm using a microplate reader.

[0117] 7. Data Processing: The formula for calculating the tyrosinase activity inhibition rate is: Y = (1 - (As - Ab) / (Ac - Ab)) * 100% (Y: tyrosinase activity inhibition rate; As: absorbance of the sample well; Ab: average absorbance of the blank group; Ac: average absorbance of the control group).

[0118] See results Figure 6 The combination of sodium mannose phosphate, nicotinamide, and hydrolyzed conchiolin significantly inhibited the activity of tyrosinase in mouse melanoma cells compared to the combination containing two other whitening agents (tranexamic acid and vitamin C).

[0119] Experimental Example 7

[0120] The effects of the compositions in comparison examples 1 to 6 on melanin production in mouse melanoma cells were tested.

[0121] 1. Cell Processing: Remove cells from the incubator, discard the old culture medium, wash once with 10 mL PBS, and discard the PBS. Add 1 mL trypsin and incubate for 1 min. After digestion, add 3 mL of complete culture medium to stop digestion. Gently pipette the cells from the bottom of the culture dish and transfer the cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant and add 1 mL of complete culture medium, gently mixing by pipetting.

[0122] 2. Cell seeding: Adjust the cell density to 11 × 10⁶ cells / year. 4 After thoroughly mixing, inoculate 2 mL of the solution into each well of a 6-well plate. Finally, incubate in an incubator for 24 hours.

[0123] 3. Sample preparation: Weigh an appropriate amount of sample into a 15ml centrifuge tube, dissolve the composition in PBS to a concentration of 100X, mix all samples thoroughly and filter through a 0.22μm filter membrane.

[0124] 4. Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table shown in Table 2.

[0125] 9. Sample addition: When the cell deposition rate in the 6-well plate reaches 40%–60%, administer the drug. Add 2 mL of culture medium containing the corresponding concentration of the sample to each well of the sample group; replace the blank group with fresh complete culture medium. After treatment, place the 6-well plate in an incubator (37℃, 5% CO2) for 24 h.

[0126] 5. Melanin Detection: Discard the supernatant, wash once with PBS, and add 200 μL of trypsin to each well for approximately 1 min. After digestion, add 1 mL of culture medium to stop the digestion, collect the cells into 1.5 mL centrifuge tubes, and centrifuge at 1000 rpm for 5 min. After centrifugation, discard the supernatant, and wash twice more with PBS. After discarding the supernatant, add 120 μL of 10% DMSO solution (prepared with 1M NaOH), vortex thoroughly, and place in an 80℃ metal bath for 30 min. Mix well by pipetting, and then transfer 100 μL of the lysis buffer to a 96-well plate. Measure the absorbance at 405 nm using a microplate reader.

[0127] See results Figure 7 The combination of sodium mannose phosphate, nicotinamide, and hydrolyzed conchiolin significantly inhibited melanin production in mouse melanoma cells compared to the combination of two other whitening agents (tranexamic acid and vitamin C).

[0128] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A whitening composition, characterized by, The active ingredients of the composition by weight parts consist of: 0.2-2 parts of sodium mannose phosphate, 100 parts of nicotinamide, and 0.8-1.2 parts of hydrolyzed conchiolin.

2. A whitening cosmetic, characterized by comprising: The active ingredients thereof include the composition of claim 1.

3. A skin lightening cosmetic product, characterized by, The active ingredients thereof include the composition of claim 1.

4. Use of the whitening composition of claim 1 for whitening for non-therapeutic purposes.

5. Use according to claim 4, characterized in that, The whitening composition is used for at least one of (1)-(3); (1) reducing or inhibiting the increase of senile melanocytes for non-therapeutic purposes; (2) maintaining the homeostasis of healthy melanocytes for non-therapeutic purposes; (3) reducing the production of melanin for non-therapeutic purposes.

6. Use of the whitening composition of claim 1 for preparing a whitening product.

7. Use according to claim 6, characterized in that, The product includes a skin care product.

8. Use according to claim 7, characterized in that, The skin care product includes any one of: a lotion, a cream, an eye cream, a mask, a moisturizing spray, a body milk, a hand cream.

9. Use according to claim 6, characterized in that, The product includes a cosmetic product.

10. Use according to claim 9, characterized in that, The cosmetic product includes any one of: a cosmetic water and a foundation.

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