Application of cyclic tetrapeptide-24 amino cyclohexane formate in preparation of whitening product

Abstract

The invention discloses application of cyclic tetrapeptide-24 amino cyclohexane formate in preparation of a whitening product, and relates to the technical field of cosmetics. The cyclic tetrapeptide-24 amino cyclohexane formate is innovatively found to be capable of inhibiting generation of melanin by inhibiting tyrosinase so as to achieve a whitening effect, has the advantages of high stability, strong activity, high permeability and the like, can realize a long-acting whitening effect only by low concentration, and is suitable for being used for a long time on sensitive skin.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to application of cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of whitening products. Background Art

[0002] Common whitening ingredients on the market, such as hydroquinone, glabridin, and vitamin C, primarily achieve their whitening effects by inhibiting tyrosinase activity, blocking melanin-promoting signals, and promoting melanin metabolism. However, these whitening agents often suffer from poor stability and permeability, leading to skin sensitivity while delivering strong results.

[0003] At present, the market is in urgent need of whitening products with superior performance.

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

[0005] The purpose of the present invention is to provide the use of cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of whitening products.

[0006] The present invention is achieved in that: In a first aspect, an embodiment of the present invention provides the use of cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of whitening products, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

[0007] In a second aspect, an embodiment of the present invention provides the use of a composition containing cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of a whitening product, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

[0008] The present invention has the following beneficial effects: The present invention innovatively discovered that cyclotetrapeptide-24 aminocyclohexanecarboxylate can inhibit the production of melanin by inhibiting tyrosinase, thereby achieving the effect of whitening; Cyclic tetrapeptide-24 aminocyclohexane carboxylate only requires low concentration to achieve long-lasting whitening effects and is suitable for long-term use on sensitive skin. Cyclic tetrapeptide-24 aminocyclohexane carboxylate has the advantages of high stability and strong activity, which can reduce the amount of preservatives and penetration enhancers added to the formula and reduce the risk of allergies; Cyclotetrapeptide-24-aminocyclohexanecarboxylate has a compact structure, can easily penetrate the stratum corneum, and has better transdermal absorption ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0010] Figure 1 This is a graph showing the melanin inhibition rate of B16-F10 cells after treatment with cyclotetrapeptide-24 aminocyclohexanecarboxylate; Figure 2 Inhibition of tyrosinase activity in B16-F10 cells after treatment with cyclic tetrapeptide-24-aminocyclohexanecarboxylate. DETAILED DESCRIPTION

[0011] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0012] Definition of noun The term "cyclotetrapeptide-24 aminocyclohexanecarboxylate" herein is named Cyclotetrapeptide-24 Aminocyclohexane, and its Chinese synonyms include cyclo(1-aminocyclohexanecarboxyl-L-arginylglycyl-L-A-aspartyl-D-phenylalanyl); cyclopentapeptide; 2-((9R,12S,18S)-9-benzylchemicalbook-18-(3-guanidinopropyl)-8,11,14,17,20-pentaoxo-7,10,13,16,19-pentaazaspiro[5.14]eicos-12-yl)acetic acid, C 28 H 40 N8O7.

[0013] The present invention innovatively discovered that cyclic tetrapeptide-24 aminocyclohexane carboxylate can inhibit the production of melanin by inhibiting tyrosinase, thereby achieving the effect of whitening; experimental verification shows that 0.04~700ppm cyclic tetrapeptide-24 aminocyclohexane carboxylate can significantly inhibit the melanin production of B16-F10 cells, and can also significantly inhibit the tyrosinase activity of B16-F10. It can be seen that it can achieve a good whitening effect under the condition of extremely low addition.

[0014] Cyclic tetrapeptide-24 aminocyclohexane carboxylate has the advantages of high stability, strong activity and high permeability. It has excellent whitening, anti-yellowing and brightening effects. It can reduce the amount of preservatives and penetration enhancers added in the formula and reduce the risk of allergies.

[0015] Technical Solution On the one hand, an embodiment of the present invention provides the use of cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of whitening products, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

[0016] In some embodiments, in the whitening product, the addition amount of the cyclotetrapeptide-24 aminocyclohexanecarboxylate is 0.04~700 ppm, specifically any one or a range between any two of 0.04 ppm, 1 ppm, 10 ppm, 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm and 700 ppm.

[0017] In some embodiments, the product comprises a cosmetic or a pharmaceutical.

[0018] On the other hand, an embodiment of the present invention further provides the use of a composition containing cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of a whitening product, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

[0019] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0020] In some embodiments, the pharmaceutically acceptable carrier includes any one or more of a pharmaceutically acceptable excipient, carrier, adjuvant, additive, surfactant, desiccant, and diluent.

[0021] Specifically, non-limiting examples of pharmaceutically acceptable carriers include lipids, sugars, amino acids, lactose, glucose, fructose, sucrose, raffinose, mannose, dextrose, trehalose, trileucine, leucine, mannitol, maltitol, xylitol, glycine, sorbitol, erythritol, phosphatidylcholine (e.g., DSPC, DSPE, etc.), calcium salts (e.g., calcium chloride, calcium sulfate), iron salts, starch, carbohydrates, cyclodextrin, cellulose, polyoxyethylene sorbitan fatty acid esters, and derivatives thereof.

[0022] Pharmaceutically acceptable carriers can be solid, semisolid or liquid.

[0023] In some embodiments, the composition further comprises: other whitening agents or cosmetic bases.

[0024] In some embodiments, the other whitening agents can be selected from existing products with whitening effects, including but not limited to phenylethylresorcinol, vitamin C derivatives (AA2G, MAP) and niacinamide.

[0025] The cosmetic base can be understood as the main component of the cosmetic, which can be obtained through conventional formulation or directly use commercially available cosmetics as the base, and mix it with the active ingredients described in the above embodiment to form the final product.

[0026] In some embodiments, in the whitening product, the added amount of the cyclotetrapeptide-24 aminocyclohexanecarboxylate is 0.04 to 700 ppm.

[0027] In some embodiments, the product comprises a cosmetic or a pharmaceutical.

[0028] In some embodiments, according to functional use, the cosmetics include cleaning products (such as facial cleansers, shower gels, and makeup remover oils), care products (such as moisturizers, essences, and sunscreens), beauty modification products (such as foundations, lipsticks, eye shadows, and blushes), hair products (such as shampoos, conditioners, and hair masks), and special function products (such as sunscreens, freckle creams, and hair removal creams).

[0029] In some embodiments, the cosmetic comprises a skin care product, and the skin care product comprises a whitening product.

[0030] In some embodiments, the cosmetic is selected from the group consisting of facial cream, lotion, essence, and cleanser.

[0031] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0032] Example 1 Synthesis method of cyclotetrapeptide-24-aminocyclohexanecarboxylate.

[0033] 1.1 Synthesis of crude cyclic peptide The target cyclic peptide synthesized is cyclotetrapeptide-24 aminocyclohexanecarboxylate (CAS No. 197172-76-2), with the INCI name CYCLOTETRAPEPTIDE-24 AMINOCYCLOHEXANE CARBOXYLATE. The target peptide was synthesized using a solid-phase synthesizer from a linear peptide, followed by cyclization by forming an amide bond between the N- and C-termini of the linear peptide.

[0034] The synthesis involved swelling 2-chlorotrityl resin in DMF, followed by sequential Fmoc deprotection using 20% ​​piperidine in DMF, and coupling of each protected amino acid (Fmoc-D-Phe-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Acc-OH) using HATU and DIPEA in DMF. After assembly of the linear peptide chain and removal of the N-terminal Fmoc group, the peptide was cleaved from the resin using 1% TFA in DCM. The resulting linear peptide, with free N- and C-terminal carboxylic acids, was then cyclized using PyBOP and DIPEA in DMF at high dilution overnight at room temperature. Finally, all side-chain protecting groups (Pbf and OtBu) were removed by treatment with a TFA:H₂O:TIPS mixture (95:2.5:2.5), and the crude cyclic peptide was precipitated with cold diethyl ether.

[0035] 1.2 Separation and purification The crude cyclic peptide was purified using reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column and a gradient of water and acetonitrile containing 0.1% TFA as the mobile phase to achieve baseline separation of the cyclic peptide from any residual linear peptide, cyclization byproducts, and other impurities. Fractions were collected based on UV absorbance at 214 or 220 nm. The collected fractions were analyzed using analytical HPLC and mass spectrometry to identify those containing the pure cyclic peptide. Pure fractions were pooled and lyophilized to obtain the purified cyclic peptide as a white solid.

[0036] Example 2 Melanin production inhibition experiment.

[0037] 2.1 Cell plating B16-F10 cells were cultured at 15 × 10 4 The cells were inoculated into 100 mm cell culture dishes at a density of 100 / mL, with an inoculation volume of 10 mL / dish. After inoculation, the culture dishes were placed in a 37°C cell culture incubator and cultured for 24 hours.

[0038] 2.2 Administration The culture dishes were divided into three groups: a blank control group, a positive control group, and 700 ppm, 100 ppm, 10 ppm, 1 ppm, and 0.04 ppm of cyclotetrapeptide-24-aminocyclohexanecarboxylate. Phenethyl resorcinol (abbreviated as "377") was used as a positive control at a final concentration of 2 ppm. After administration, the cells were cultured in a 37°C cell culture incubator for 24 hours.

[0039] 2.3 Melanin content detection The cells were digested with trypsin and collected by centrifugation. The supernatant was discarded and washed twice with PBS. The supernatant was discarded again and the cells were lysed by adding 300 μL of 1 M NaOH containing 10% DMSO. The cells were placed in an 80°C water bath for 30 minutes. After lysis, 200 μL of cell lysate was transferred to a 96-well plate and the absorbance at 405 nm was read using a microplate reader. The absorbance was converted to melanin content, with the melanin content of the control group set as 100%.

[0040] 2.4 Data Analysis After the test, the obtained data was analyzed. The calculation formula of melanin inhibition rate is as follows: ; Where, As: absorbance of the sample group; Ac: average absorbance of the blank control group.

[0041] The independent sample T test was used to analyze the significant differences between the sample treatment group and the control group. P <0.05 was considered statistically significant. P <0.05,** P <0.01.

[0042] 2.5 Experimental Results like Figure 1 As shown, the melanin production inhibition rates of 700ppm, 100ppm, 10ppm, 1ppm and 0.04ppm cyclotetrapeptide-24aminocyclohexanecarboxylate on B16-F10 cells were 39.20%, 47.12%, 29.63%, 13.63% and 12.42%, respectively, indicating that cyclotetrapeptide-24aminocyclohexanecarboxylate has good whitening effect.

[0043] Example 3 Tyrosinase activity inhibition assay in B16F10 cells.

[0044] 3.1 Cell seeding B16F10 cells (mouse melanoma cells) grown in DMEM complete medium were cultured at a density of 6 × 10 4 Cells were seeded into 96-well cell culture plates at a density of 100 cells / well, with 2 mL per well. After seeding, the 96-well plates were placed in a cell culture incubator for 24 h.

[0045] 3.2 Administration The experiment was divided into three groups: a blank control group, 700 ppm, 100 ppm, 10 ppm, 1 ppm, and 0.04 ppm cyclotetrapeptide-24-aminocyclohexanecarboxylate groups, and a positive control group. Six replicates were set up in each group. An equal volume of PBS was added to the blank control group. Phenethyl resorcinol (abbreviated as "377") was used as a positive control at a final concentration of 2 ppm. After dosing, the 96-well plates were placed in a cell culture incubator for 24 hours.

[0046] 3.3 Determination of tyrosinase activity After 24 hours of incubation, the supernatant was discarded, and the cells were washed three times with PBS. 80 μL of 1% Triton X-100 solution was added to each well and the cells were immediately frozen at -81°C for 30 minutes. The cells were then thawed at room temperature to completely rupture the cells. After prewarming at 37°C for 5 minutes, 20 μL of 1% L-DOPA solution was added. The cells were incubated at 37°C for 2 hours, and the absorbance was measured at 490 nm using a microplate reader.

[0047] 3.4 Results Statistics and Analysis After the test, the obtained data was analyzed. The calculation formula of tyrosinase activity inhibition rate is as follows: ; Where, As: absorbance of the sample group; Ac: average absorbance of the blank control group.

[0048] The independent sample T test was used to analyze the significant differences between the sample treatment group and the control group. P <0.05 was considered statistically significant. P <0.05, **P<0.01.

[0049] 3.5 Experimental Results like Figure 2 As shown, the tyrosinase activity inhibition rates of 700ppm, 100ppm, 10ppm, 1ppm, and 0.04ppm cyclotetrapeptide-24aminocyclohexanecarboxylate were 43.19%, 40.07%, 34.33%, 25.51%, and 10.13%, respectively, indicating that cyclotetrapeptide-24aminocyclohexanecarboxylate can achieve a whitening effect by inhibiting tyrosinase activity.

[0050] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of whitening products, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

2. The use according to claim 1, characterized in that In the whitening product, the added amount of the cyclotetrapeptide-24 aminocyclohexanecarboxylate is 0.04-700 ppm.

3. The use according to any one of claims 1 to 2, characterized in that The product includes a cosmetic or a medicine.

4. Use of a composition containing cyclotetrapeptide-24-aminocyclohexanecarboxylate in the preparation of a whitening product, wherein the CAS number of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 197172-76-2.

5. The use according to claim 4, characterized in that The composition also includes a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that The pharmaceutically acceptable carrier includes any one or more of pharmaceutically acceptable excipients, carriers, adjuvants, additives, surfactants, desiccants and diluents.

7. The use according to claim 6, characterized in that The composition further comprises: other whitening agents or cosmetic bases.

8. The use according to claim 6, characterized in that In the whitening product, the added amount of the cyclotetrapeptide-24-aminocyclohexanecarboxylate is 0.04~700 ppm.

9. The use according to any one of claims 5 to 8, characterized in that The product includes a cosmetic or a medicine.

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