An active peptide with anti-wrinkle function and its application in cosmetics

Abstract

The present invention relates to the technical field of active peptides, and provides an active peptide with anti-wrinkle function and its application in cosmetics. The present invention first chloromethylates the benzene ring of dibenzo-18-crown-6 with 1,4-dichloromethoxybutane, and then grafts the chloromethylated dibenzo-18-crown-6 onto the side chain of palmitoyl pentapeptide-4 to obtain a new active peptide. Compared with unimproved palmitoyl pentapeptide-4, the active peptide prepared by the present invention can play a better role in stimulating collagen production and reducing collagen degradation, thereby increasing the collagen content in the dermis and providing better elasticity and support for the skin. Therefore, cosmetics adding the active peptide can play a better anti-wrinkle effect.

Description

Technical Field

[0001] The invention relates to the technical field of active peptides and provides an active peptide with anti-wrinkle function and application thereof in cosmetics. Background Art

[0002] The skin is the largest organ in the human body. Since the skin is in direct contact with the external environment, it reacts quickly to external changes. With age, the skin gradually ages under the combined effects of various exogenous factors (such as ultraviolet radiation, air pollution, temperature changes, etc.) and endogenous factors (such as genetics, hormone stimulation, etc.), which is mainly manifested in the formation of wrinkles and reduced elasticity.

[0003] The most relevant to wrinkles is the extracellular matrix of the dermis, which is composed of collagen, elastin, fibrin, proteoglycan complexes, etc., which can give the skin elasticity and support. Adding collagen, elastin, etc. to cosmetics can achieve certain anti-wrinkle and firming effects, but they are large molecular proteins with poor transdermal absorption and are difficult to penetrate the stratum corneum of the skin and be absorbed.

[0004] Active peptides refer to peptides that can play special functions in organisms. They can be as small as two amino acids or as large as hundreds of amino acids. Their structure size is between amino acids and proteins. In the field of beauty and skin care, different active peptides can play a variety of roles such as whitening, anti-oxidation, anti-aging, antibacterial, and repair. The development of active peptides with good anti-wrinkle function has broad application prospects. Summary of the invention

[0005] The present invention provides an active peptide with anti-wrinkle function and application thereof in cosmetics, in order to obtain good anti-wrinkle effect.

[0006] The specific technical solutions involved in the present invention are as follows:

[0007] First, the present invention provides a method for preparing an active peptide with anti-wrinkle function, the preparation steps of the active peptide are:

[0008] (1) Dissolve dibenzo-18-crown-6 in chloroform, then dropwise add a mixed solution of 1,4-dichloromethoxybutane and anhydrous tin tetrachloride, and react at room temperature with stirring. After the reaction is completed, add dilute hydrochloric acid, stir, separate the organic phase after oil and water phase separation, wash with deionized water, concentrate by rotary evaporation, and dry in vacuum to obtain chloromethylated dibenzo-18-crown-6;

[0009] (2) Dissolve the chloromethylated dibenzo-18-crown-6 in N,N-dimethylformamide, add palmitoyl pentapeptide-4 and sodium carbonate, heat and stir to react, add dilute hydrochloric acid after the reaction is completed, stir, separate the organic phase after oil and water phase separation, wash with deionized water, concentrate by rotary evaporation, and vacuum dry to obtain the active peptide with anti-wrinkle function.

[0010] Step (1) uses 1,4-dichloromethoxybutane to chloromethylate the benzene ring of dibenzo-18-crown-6. The molar ratio of dibenzo-18-crown-6 to 1,4-dichloromethoxybutane is 2:1. After the reaction, a chloromethyl group is attached to each dibenzo-18-crown-6 molecule.

[0011] Preferably, in step (1), the reaction time is 100-150 min.

[0012] Preferably, in step (1), the amount of dibenzo-18-crown-6 added to chloroform is 15-25 g / L; and the amount of anhydrous tin tetrachloride added is 4-6% of the mass of 1,4-dichloromethoxybutane.

[0013] Further preferably, in step (1), the concentration of dilute hydrochloric acid is 1 mol / L, and the volume ratio of dilute hydrochloric acid to chloroform is 0.8-1:1.

[0014] Step (2) uses chloromethylated dibenzo-18-crown-6 to react with palmitoyl pentapeptide-4. The molar ratio of chloromethylated dibenzo-18-crown-6 to palmitoyl pentapeptide-4 is 1:1. After the reaction, a benzocrown structure is connected to each palmitoyl pentapeptide-4 molecule. The molecular structure of palmitoyl pentapeptide-4 contains two amino groups and three hydroxyl groups. Since the amino group has a stronger reaction ability with chloromethyl than with hydroxyl groups, the chloromethylated dibenzo-18-crown-6 is connected by reacting with the amino group of palmitoyl pentapeptide-4. For the two amino groups, since one of the amino groups is closer to the long-chain structure of palmitamide and the reaction steric hindrance is greater, the other amino group is more easily connected by reacting with the chloromethyl group. The structural formula of the product is shown in the attached figure. Figure 1 .

[0015] Preferably, in step (2), the reaction temperature is 68-72°C and the reaction time is 8-10 hours.

[0016] Preferably, in step (2), the amount of chloromethylated dibenzo-18-crown ether-6 added to N,N-dimethylformamide is 4-6 g / L; and the amount of sodium carbonate added is 30-40% of the mass of palmitoyl pentapeptide-4.

[0017] Further preferably, in step (2), the concentration of dilute hydrochloric acid is 1 mol / L, and the volume ratio of dilute hydrochloric acid to N,N-dimethylformamide is 0.8-1:1.

[0018] Secondly, the present invention provides an active peptide with anti-wrinkle function prepared by the above preparation method.

[0019] As is known, palmitoyl pentapeptide-4 (Pal-KTTKS for short) is a small molecule polypeptide formed by connecting collagen I fragments and palmitic acid through amide bonds, and the peptide sequence is Pal-Lys-Thr-Thr-Lys-Ser-OH. Palmitoyl pentapeptide-4 is a signal peptide that can stimulate fibroblasts to produce collagen, thereby playing an anti-wrinkle role. Existing studies have shown that KTTKS is the smallest peptide chain required to stimulate fibroblasts to produce collagen. KTTKS is different from other substances that act on the stratum corneum or epidermis, and its location of action is in the dermis. In order to promote KTTKS to reach the dermis, palmitic acid is grafted on the end of KTTKS. Since palmitic acid is one of the main components of stratum corneum lipids, it can destroy the accumulation of intercellular lipids in the skin layer and improve the skin permeability of KTTKS.

[0020] However, the use of palmitoyl pentapeptide-4 as an anti-wrinkle peptide also has certain defects. Due to the presence of aminopeptidase in the skin, which is an exoproteinase that can selectively remove amino acid residues from the N-terminus of the peptide, the presence of aminopeptidase causes Pal-KTTKS to be quickly enzymatically hydrolyzed after entering the skin. Part of KTTKS cannot reach the deep layer of the skin under the action of Pal, and the effective amount that can reach the dermis to exert its effect is low, which reduces the effect of use.

[0021] In view of this, the present invention has made the above-mentioned improvements to palmitoyl pentapeptide-4, and the improved active peptide side chain contains a benzocrown ether structure. Its advantages in anti-wrinkle are:

[0022] First, it is known that aminopeptidase requires Zn 2+ Participation, catalytic activity has Zn 2+ The catalytic center of aminopeptidase consists of two lipophilic pockets and a Zn 2+ The binding region, structurally, contains a lipophilic group (bound to the lipophilic pocket) and Zn 2+ Chelating group (with Zn 2+ The active peptide prepared by the present invention contains a long alkyl chain (from the palmitoyl part) which can bind to the lipophilic pocket of the aminopeptidase, change the spatial structure of the enzyme and reduce the activity. On the other hand, it contains a dibenzo-18-crown-6 structure, which has an inhibitory effect on the Zn binding of the aminopeptidase. 2+ Has good complexing ability and can reduce Zn 2+The active peptide prepared by the present invention can inhibit the activity of aminopeptidase by its own structure, prevent itself from being prematurely degraded by aminopeptidase in the skin, thereby increasing its effective amount reaching the dermis and stimulating fibroblasts to produce more collagen.

[0023] In addition, matrix metalloproteinases can degrade collagen in the dermis, destroying the structure of the extracellular matrix and making the skin no longer elastic and supportive. Inhibiting the activity of matrix metalloproteinases can prevent the formation of skin wrinkles. Unlike aminopeptidases, matrix metalloproteinases are endoproteinases. Similar to aminopeptidases, matrix metalloproteinases require metal ions (such as Zn 2+ , Ca 2+ ) as an auxiliary. Therefore, the active peptide prepared by the present invention can also form a complex with the metal ions in the active center of the matrix metalloproteinase by utilizing its dibenzo-18-crown ether-6 structure, and compete with the interaction between the metal ions and the substrate, thereby reducing the degradation of collagen in the dermis.

[0024] Thirdly, the present invention provides the application of the above active peptides with anti-wrinkle function in cosmetics.

[0025] Preferably, the cosmetic is in the form of an emulsion, gel or cream. Since the above active peptides can stimulate collagen production and reduce collagen degradation, after being added to cosmetics, skin elasticity can be significantly improved and skin wrinkles can be reduced.

[0026] The present invention provides an active peptide with anti-wrinkle function and its application in cosmetics, and its beneficial effect is that: the present invention improves palmitoyl pentapeptide-4, introduces a benzocrown ether structure on its side chain, and obtains a new active peptide. The active peptide can not only rely on its own structure to inhibit the activity of aminopeptidase, reduce the speed of its own degradation by aminopeptidase, ensure that it reaches the dermis and stimulates fibroblasts to produce collagen, that is, overcomes the defect that palmitoyl pentapeptide-4 is easily degraded by aminopeptidase and reduces the use effect; moreover, the active peptide can inhibit the activity of matrix metalloproteinases and reduce the degradation of collagen in the dermis by matrix metalloproteinases. That is to say, compared with unimproved palmitoyl pentapeptide-4, the active peptide can play a better role in stimulating collagen production and reducing collagen degradation, thereby increasing the collagen content in the dermis and providing better elasticity and support for the skin. Therefore, cosmetics added with the active peptide can have a better anti-wrinkle effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the structural formula of the active peptide prepared by the present invention. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below through specific embodiments, but this should not be construed as limiting the scope of the present invention to the following examples. Without departing from the above-mentioned method concept of the present invention, various substitutions or changes made according to common technical knowledge and conventional means in the art should be included in the scope of the present invention.

[0029] Example 1

[0030] (1) Dissolve dibenzo-18-crown-6 in chloroform, then dropwise add a mixed solution of 1,4-dichloromethoxybutane and anhydrous tin tetrachloride, and react at room temperature for 120 minutes with stirring. After the reaction is completed, add 1 mol / L dilute hydrochloric acid (equal volume to chloroform), stir, separate the organic phase after oil-water phase separation, wash with deionized water, concentrate by rotary evaporation, and dry in vacuum to obtain chloromethylated dibenzo-18-crown-6; the molar ratio of dibenzo-18-crown-6 to 1,4-dichloromethoxybutane is 2:1; the amount of dibenzo-18-crown-6 added to chloroform is 20 g / L; the amount of anhydrous tin tetrachloride added is 5% of the mass of 1,4-dichloromethoxybutane;

[0031] (2) Dissolve chloromethylated dibenzo-18-crown-6 in N,N-dimethylformamide, add palmitoyl pentapeptide-4 and sodium carbonate, heat to 70°C and stir for 9 hours. After the reaction, add 1 mol / L dilute hydrochloric acid (equal volume to N,N-dimethylformamide), stir, separate the oil and water phases, and then separate the organic phase. Wash with deionized water, concentrate by rotary evaporation, and dry in vacuum to obtain the active peptide with anti-wrinkle function. The molar ratio of chloromethylated dibenzo-18-crown-6 to palmitoyl pentapeptide-4 is 1:1. The amount of chloromethylated dibenzo-18-crown-6 added to N,N-dimethylformamide is 5 g / L. The amount of sodium carbonate added is 35% of the mass of palmitoyl pentapeptide-4.

[0032] Comparative Example 1

[0033] Unmodified Palmitoyl Pentapeptide-4.

[0034] Example 2

[0035] A gel cosmetic was prepared with the following formula: 0.2 g of the active peptide of Example 1, 1 g of carbomer, 15 g of glycerol, and 84 g of water.

[0036] Preparation process: add carbomer to glycerin to fully moisten it, add half of water to make the carbomer fully swell and form a hydrated carbomer colloid; add the active peptide of Example 1 to the other half of the water to form a stable solution; slowly add the stable solution to the carbomer colloid, knead evenly, and adjust the pH value to 6.

[0037] Comparative Example 2

[0038] A gel cosmetic was prepared, the formula of which was: 0.2 g of palmitoyl pentapeptide-4 of Comparative Example 1, 1 g of carbomer, 15 g of glycerin, and 84 g of water.

[0039] Preparation process: add carbomer to glycerin, fully moisten it, add half of water to make the carbomer fully swell and form a hydrated carbomer colloid; add palmitoyl pentapeptide-4 of comparative example 1 to the other half of the water to form a stable solution; slowly add the stable solution to the carbomer colloid, knead evenly, and adjust the pH value to 6.

[0040] Example 3

[0041] A gel cosmetic was prepared with the following formula: 0.4 g of the active peptide of Example 1, 1 g of carbomer, 15 g of glycerin, and 84 g of water.

[0042] The preparation process is the same as Example 2.

[0043] Comparative Example 3

[0044] A gel cosmetic was prepared, the formula of which was: 0.4 g of palmitoyl pentapeptide-4 of Comparative Example 1, 1 g of carbomer, 15 g of glycerin, and 84 g of water.

[0045] The preparation process is the same as that of Comparative Example 2.

[0046] Example 4

[0047] A gel cosmetic was prepared with the following formula: 0.6 g of the active peptide of Example 1, 1 g of carbomer, 15 g of glycerin, and 84 g of water.

[0048] The preparation process is the same as Example 2.

[0049] Comparative Example 4

[0050] A gel cosmetic was prepared, the formula of which was: 0.6 g of palmitoyl pentapeptide-4 of Comparative Example 1, 1 g of carbomer, 15 g of glycerin, and 84 g of water.

[0051] The preparation process is the same as that of Comparative Example 2.

[0052] Performance Test:

[0053] (1) Take fresh abdominal skin of healthy SD rats, weigh 5 g of skin tissue, cut it into pieces, add it to 100 mL of ultrapure water, stir it for 2 h, filter out large pieces of tissue, centrifuge, take the upper solution and freeze-dry it to obtain enzyme solution freeze-dried powder; thaw the freeze-dried powder at room temperature, divide it into two parts, add 0.01 mol of the active peptide of Example 1 to one part, and add 0.01 mol of palmitoyl pentapeptide-4 of Comparative Example 1 to the other part; place it in a shaker, set the temperature to 37°C and the speed to 300 rpm; take 1 mL of sample at 1 h, 2 h, 3 h, 4 h, and 5 h, respectively, break the emulsion with Triton (10 wt%) and adjust the volume to 10 mL, enter HPLC analysis, and obtain the cumulative degradation rate of the polypeptide at each time point.

[0054] The test results are shown in Table 1. As can be seen from Table 1, the unmodified palmitoyl pentapeptide-4 is degraded quickly in the enzyme solution, while the active peptide of the present invention can inhibit the activity of aminopeptidase by its own structure, so its own degradation rate is significantly reduced.

[0055] Table 1:

[0056]

[0057] (2) The active peptide of Example 1 and the palmitoyl pentapeptide-4 of Comparative Example 1 were added to sterile distilled water to prepare a 2.5 mmol / L polypeptide solution; matrix metalloproteinase (MMP-1) and 1 μL of the polypeptide solution were added to a 96-well ELISA plate (the enzyme amount was 500 U / g, and sterile distilled water was set to replace the polypeptide solution as a blank control sample), and a buffer solution (50 mmol / L HEPES, 0.2 mol / L NaCl, 10 mmol / L CaCl2, 20 μmol / L ZnSO4, 0.05% Brij-35) was added to a volume of 50 μL, and the plate was placed in a 37°C incubator for 30 min, and 50 μL of a buffer solution containing 0.2 μg of a fluorescent substrate (purchased from Sigma, USA) was added, and the plate was immediately stained with Bio-Red Detection was performed using a FLx-800 fluorescence microplate reader with an excitation wavelength of 460 nm and an emission wavelength of 520 nm. The detection was performed for 10 min to obtain the change in fluorescence intensity. The inhibition percentage of matrix metalloproteinases was calculated according to the following formula: inhibition percentage = (1-Vi / V0) × 100%, where Vi is the change in fluorescence intensity of the polypeptide solution sample, and V0 is the change in fluorescence intensity of the blank control sample.

[0058] Test results: The inhibition percentage of the active peptide of Example 1 is 37.3%, and the inhibition percentage of palmitoyl pentapeptide-4 of Comparative Example 1 is 6.2%. It can be seen that the unimproved palmitoyl pentapeptide-4 has a poor inhibitory effect on the activity of matrix metalloproteinases, while the active peptide of the present invention can form a complex with the metal ion of the active center of the matrix metalloproteinase by using the benzocrown ether structure, thereby significantly inhibiting the activity of the matrix metalloproteinase.

[0059] (3) 120 healthy women (divided into 3 groups, 40 in each group) with mild or above eye wrinkles around the eyes were selected as subjects. The test site was the skin around the eyes. The gel cosmetics of Examples 2-4 were used on the left eye of each group of subjects, and the gel cosmetics of Comparative Examples 2-4 were used on the right eye of each group of subjects. The use method was: twice a day, after cleansing in the morning and evening, apply an appropriate amount of the product to the corresponding skin surface around the eyes, and gently massage along the skin around the eyes with the fingertips until it is completely absorbed. If there is an adverse reaction on the skin during the test, stop using it immediately. Before use, after 4 weeks of use, and after 12 weeks of use, use Viscia-CR facial image analyzer to collect facial skin images to obtain the crow's feet area, and the data of the subjects in the same group were averaged; the reduction rate of the crow's feet area was calculated according to the following formula: Reduction rate = (1-Si / S0) × 100%, where Si is the crow's feet area after use, and S0 is the crow's feet area before use.

[0060] The test results are shown in Table 2. It can be seen from Table 2 that compared with the cosmetics containing the palmitoyl pentapeptide-4 of Comparative Example 1, the cosmetics containing the active peptide of Example 1 have better anti-wrinkle effects, which are due to: first, the active peptide of the present invention can inhibit the activity of aminopeptidase by its own structure, and its own degradation rate is slower after entering the skin, and the effective amount reaching the dermis is more, which can stimulate the production of more collagen; second, after the active peptide of the present invention reaches the dermis, it has an active inhibitory effect on matrix metalloproteinases, which can reduce the degradation of collagen in the dermis.

[0061] Table 2:

[0062]

Claims

1. A method for preparing an active peptide with anti-wrinkle function, characterized in that: The preparation steps of the active peptide are: (1) Dissolve dibenzo-18-crown-6 in chloroform, then dropwise add a mixed solution of 1,4-dichloromethoxybutane and anhydrous tin tetrachloride, and react at room temperature with stirring. After the reaction is completed, add dilute hydrochloric acid, stir, separate the organic phase after oil and water phase separation, wash with deionized water, concentrate by rotary evaporation, and dry in vacuum to obtain chloromethylated dibenzo-18-crown-6; (2) dissolving chloromethylated dibenzo-18-crown-6 in N,N-dimethylformamide, adding palmitoyl pentapeptide-4 and sodium carbonate, heating and stirring for reaction, adding dilute hydrochloric acid after the reaction, stirring, separating the organic phase after oil-water phase separation, washing with deionized water, concentrating by rotary evaporation, and vacuum drying to obtain the active peptide with anti-wrinkle function; In step (1), the molar ratio of dibenzo-18-crown-6-yl and 1,4-dichloromethoxybutane is 2:1; In step (2), the molar ratio of chloromethylated dibenzo-18-crown-6 to palmitoyl pentapeptide-4 is 1:1; In step (2), the reaction temperature is 68-72°C and the reaction time is 8-10 hours.

2. The method for preparing an active peptide with anti-wrinkle function according to claim 1, characterized in that: In step (1), the reaction time is 100-150 min.

3. The method for preparing an active peptide with anti-wrinkle function according to claim 1, characterized in that: In step (1), the amount of dibenzo-18-crown-6 added to chloroform is 15-25 g / L; the amount of anhydrous tin tetrachloride added is 4-6% of the mass of 1,4-dichloromethoxybutane.

4. The method for preparing an active peptide with anti-wrinkle function according to claim 1, characterized in that: In step (1), the concentration of dilute hydrochloric acid is 1 mol / L, and the volume ratio of dilute hydrochloric acid to chloroform is 0.8-1:

1.

5. The method for preparing an active peptide with anti-wrinkle function according to claim 1, characterized in that: In step (2), the amount of chloromethylated dibenzo-18-crown ether-6 added to N,N-dimethylformamide is 4-6 g / L; the amount of sodium carbonate added is 30-40% of the mass of palmitoyl pentapeptide-4.

6. The method for preparing an active peptide with anti-wrinkle function according to claim 1, characterized in that: In step (2), the concentration of dilute hydrochloric acid is 1 mol / L, and the volume ratio of dilute hydrochloric acid to N,N-dimethylformamide is 0.8-1:

1.

7. The active peptide with anti-wrinkle function prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the active peptide with anti-wrinkle function according to claim 7 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that: The cosmetic is in the form of emulsion, gel or paste.

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