Peptide composition for brightening skin as well as preparation method and application method thereof

Abstract

The invention discloses a peptide composition for brightening skin as well as a preparation method and an application method thereof, and relates to the technical field of cosmetics. The invention discloses a preparation method of a peptide composition for brightening skin, which comprises the following steps: mixing active peptide microcapsules, 1, 3-propylene glycol, caprylyl glycol, ethylhexylglycerin and water to obtain the peptide composition; the active peptide microcapsules are obtained by carrying out loading treatment on dipeptide-15 and oligopeptide-4; the dipeptide-15 and the oligopeptide-4 have a synergistic effect to serve as an active peptide component of the peptide composition, so that the activity of skin cells is effectively improved, pigmentation of face skin is remarkably reduced, and the effect of brightening the skin is achieved. The active peptide component is loaded, so that the transdermal absorption rate and the stability of the active peptide component are effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and particularly relates to a peptide composition for brightening skin, a preparation method thereof, and an application method thereof. Background Art

[0002] Except for anaerobic bacteria, most organisms use oxygen as the terminal electron acceptor to oxidize various metabolic fuels, so as to store energy for the survival of various organisms. This process occurs in the mitochondria of eukaryotic cells. In addition to synthesizing adenosine triphosphate (ATP), mitochondria also have a variety of extremely important functions, including the generation of reactive oxygen species (ROS), the regulation of redox potential and the conduction of cellular redox signals, as well as the regulation of apoptosis and gene expression. Normal cells can produce ROS genes. Under physiological conditions, the mitochondrial respiratory chain is the main source of ROS. Mitochondria are not only the basic sites for generating ROS, but also the main targets of ROS damage. Increasing ROS can attack membrane lipids and proteins, including various enzymes and DNA, and can deteriorate the functions of cells.

[0003] The ROS-mitochondrial damage cycle is an important cause of skin aging. The harmful effects and accumulation of ROS in life have always been regarded as one of the important reasons for the aging of the body. Mitochondria in aging tissues show a decline in respiratory capacity and insufficient biochemical functions. A large number of studies have shown that biochemical changes in the electron transport chain cause an increase in RO generation and an increase in lipid peroxidation damage, thus causing mitochondrial aging, and mitochondrial aging causes the aging of life. According to the "free radical theory", the aging of cells and tissues is the result of the attack of free radicals. In human cells, mitochondria are the sites of multiple redox reactions that generate ATP energy, so they are the main source of ROS in cells. Under normal physiological conditions, about 0.4% to 4% of oxygen is reduced to ROS. Changes in the internal and external environment lead to mitochondrial dysfunction, resulting in a decrease in ATP production and an increase in ROS production, which will further damage mitochondria. This vicious cycle will lead to serious cell damage and accelerate the aging process.

[0004] Peptides are the general term for linear or cyclic peptides composed of amino caproic acids through peptide bonds in different compositions and arrangements. Bioactive peptides generally refer to the general term for peptides that have certain physiological functions in addition to nutritional functions, and play important roles in beauty, regulating gastrointestinal motility, immune regulation, anti-hypertension, antibacterial, anti-thrombosis, antioxidant, promoting the absorption of mineral elements, etc. Polypeptide substances, including collagen, elastin, palmitoyl peptide, copper peptide, carnosine, etc., are the main bioactive peptides applied to cosmetics and are used as anti-aging agents for the body. However, such protein molecules are large in volume and usually cannot be directly absorbed by the skin. Summary of the Invention

[0005] The object of the present invention is to provide a peptide composition for brightening the skin, its preparation method and application method, and solve the following technical problems:

[0006] For existing common polypeptide substances, the protein molecules are large in volume and difficult to be directly absorbed by the skin.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] A preparation method of a peptide composition for brightening the skin, comprising the following steps: mixing energizing peptide microcapsules, 1,3-propanediol, caprylyl glycol, ethylhexylglycerin, and water to obtain a peptide composition; the energizing peptide microcapsules are obtained by loading oligopeptide-4 and dipeptide-15 as peptide active substances;

[0009] The preparation method of the energizing peptide microcapsules comprises the following steps:

[0010] S1: Under nitrogen protection, add monomethoxypolyethylene glycol, ε-caprolactone, and stannous octoate into a reaction kettle and disperse evenly, control the temperature at 70 - 80 °C, keep warm and react for 18 - 36 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0011] S2: After blending the copolymer, histidine, N-isopropylacrylamide, and N,N-dimethylformamide, blend EDC and NHS and add them, control the temperature at 55 - 65 °C, keep warm and react for 8 - 16 h, dialyze to remove unreacted monomers, and dry to obtain a functionalized copolymer;

[0012] S3: Add the functionalized copolymer, oligopeptide-4, and tetrahydrofuran into a reaction kettle for ultrasonic emulsification, add the dipeptide-15-sodium alginate aqueous solution into the reaction kettle and disperse evenly, remove tetrahydrofuran, concentrate to obtain a drug-loaded micelle with a solid content of 10 - 20%;

[0013] S4: Blend α-lipoic acid and absolute ethanol, add the drug-loaded micelle, control the temperature at 40 - 50 °C, keep warm for 3 - 6 h under stirring conditions, ultrafilter and concentrate to obtain energizing peptide microcapsules.

[0014] As a further scheme of the present invention: oligopeptide-4 accounts for 0.01 - 0.05% of the total mass of the peptide composition; 1,3-propanediol accounts for 1.0 - 1.5% of the total mass of the peptide composition; caprylyl glycol accounts for 0.2 - 0.5% of the total mass of the peptide composition; ethylhexylglycerin accounts for 0.05 - 0.08% of the total mass of the peptide composition.

[0015] As a further scheme of the present invention: dipeptide-15 accounts for 3 - 8% of the total mass of the peptide composition.

[0016] As a further solution of the present invention: when dipeptide-15 accounts for 8-13% of the total mass of the peptide composition; the peptide composition further includes butylene glycol; and the butylene glycol accounts for 8-13% of the total mass of the peptide composition.

[0017] As a further solution of the present invention: in S1, the addition ratio of monomethoxypolyethylene glycol, ε-caprolactone, and stannous octoate is 100 g: 200-300 g: 0.15-0.25 g.

[0018] As a further solution of the present invention: in S2, the addition ratio of the copolymer, histidine, N-isopropylacrylamide, N,N-dimethylformamide, EDC, and NHS is 100 g: 8-12 g: 4-6 g: 1000-2000 mL: 10-20 g: 5-10 g.

[0019] As a further solution of the present invention: in the dipeptide-15-sodium alginate aqueous solution in S3, it includes 5-15 wt% of dipeptide-15 and 0.5-1 wt% of sodium alginate;

[0020] The addition ratio of the functionalized copolymer, oligopeptide-4, tetrahydrofuran, and dipeptide-15-sodium alginate aqueous solution is 10 g: 0.01-0.1 g: 100-200 mL: 40 mL.

[0021] As a further solution of the present invention: in S4, the addition ratio of α-lipoic acid, absolute ethanol, and drug-loaded micelles is 0.5-2 g: 100-200 mL: 100 g.

[0022] A peptide composition for brightening the skin, which is prepared by any of the above preparation methods.

[0023] The application method of the above peptide composition, where the peptide composition is applied in skin care products, and the peptide composition accounts for 0.1-20 wt% of the mass of the skin care products.

[0024] As a further solution of the present invention: the skin care product is any one of leave-on skin care products and rinse-off skin care products.

[0025] The beneficial effects of the present invention:

[0026] (1) In the present application, oligopeptide-4 and dipeptide-15 are added to the peptide composition to act synergistically, enhancing the vitality of skin cells and achieving the effect of brightening the skin. Experimental data shows that the peptide composition of the present invention can promote the proliferation of human skin fibroblasts, promote the release of cellular ATP, effectively reduce the increase in intracellular ROS caused by oxidative damage, exhibit significant antioxidant activity, and can significantly reduce pigment deposition on the facial skin and brighten the skin color.

[0027] (2) In this application, stannous octoate is used as a catalyst to initiate the ring-opening polymerization of ε-caprolactone (CL) through monomethoxy polyethylene glycol to prepare a copolymer; and the copolymer is grafted with histidine, and N-isopropylacrylamide is also introduced during the grafting process to prepare a functional copolymer; then, the functional copolymer is used to load oligopeptide-4 and dipeptide-15 to obtain a drug-loaded micelle; among them, the PCL segment of the functional copolymer can effectively encapsulate the hydrophobic oligopeptide-4 and stabilize its α-helix structure through hydrophobic interaction; the PEG segment of the functional copolymer forms a hydrogen bond network with dipeptide-15 as a hydrophilic outer shell to load dipeptide-15; finally, α-lipoic acid molecules are covalently bonded to provide antioxidant protection and form a disulfide bond cross-linking network to obtain an active peptide microcapsule.

[0028] In this application, the copolymer is used as the core skeleton, and histidine and N-isopropylacrylamide are used for functional modification to endow the material with pH-responsive units and temperature-responsive units, effectively realizing controlled release; during the preparation of the active peptide microcapsule, α-lipoic acid and sodium alginate are covalently bonded to form an antioxidant-humidity retention double network, effectively inhibiting the hydrolysis of oligopeptide-4 and dipeptide-15; improving the stability of peptide active substances. In this application, oligopeptide-4 and dipeptide-15 are processed to obtain an active peptide microcapsule, which not only effectively improves the effect of the active peptide in penetrating the skin barrier and enhances the skin absorption effect; but also effectively improves the stability of the active peptide.

[0029] (3) The present application adds 1,3-propylene glycol, caprylyl glycol and ethylhexylglycerin to the peptide composition; among them, 1,3-propylene glycol, as a small molecule polyol, helps dissolve peptides and lock in moisture, reduces the stickiness of the formula, and improves the freshness of the skin. It also combines with peptides through hydrogen bonds to reduce the hydrolysis or oxidative degradation of peptide chains and prolong the active shelf life. 1,3-propylene glycol can also slightly soften the stratum corneum, assist in the transdermal absorption of peptides, and is less irritating than traditional propylene glycol (1,2-propylene glycol). Caprylyl glycol, as a polyol preservative, inhibits the growth of bacteria and fungi (especially effective against Gram-positive bacteria) and reduces the interference of traditional preservatives (such as parabens) on peptides. Caprylyl glycol works synergistically with thiol groups (such as glutathione) in peptides to scavenge free radicals and protect the integrity of peptide structures. Caprylyl glycol has light emollient properties, improves the extensibility of the formula, and avoids the "mud rubbing" phenomenon of peptide essences. Ethylhexylglycerin can enhance the antibacterial effect of caprylyl glycol, especially for stubborn microorganisms such as Pseudomonas aeruginosa, and reduce the risk of peptide contamination. It also increases the residence time and efficiency of peptides in the epidermis by enhancing the hydration of the stratum corneum. As a chelating agent precursor, ethylhexylglycerin can indirectly complex metal ions (such as iron and copper) to prevent metal-catalyzed peptide oxidation. The synergistic combination of 1,3-propylene glycol, caprylyl glycol and ethylhexylglycerin can construct a "traditional preservative-free" system (especially avoiding phenoxyethanol), reduce irritation to sensitive skin, avoid destroying the activity of peptides, and increase the transdermal penetration of small molecule peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below in conjunction with the accompanying drawings.

[0031] Figure 1 This is the fluorescent staining diagram of the antioxidant activity test of this application;

[0032] Figure 2 This is a staining diagram of the collagen regeneration test in this application. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1 The preparation method of active peptide microcapsules comprises the following steps:

[0035] S1: Under nitrogen protection, add 100 g of monomethoxypolyethylene glycol (MW = 2000), 200 g of ε-caprolactone, and 0.15 g of stannous octoate into a reaction kettle, disperse them evenly, control the temperature at 70 °C, keep the temperature for reaction for 18 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0036] S2: After blending 100 g of copolymer, 8 g of histidine, 4 g of N-isopropylacrylamide, and 100 mL of N,N-dimethylformamide, blend 10 g of EDC and 5 g of NHS and add them, control the temperature at 55 °C, keep the temperature for reaction for 8 h, dialyze to remove unreacted monomers, and dry to obtain a functionalized copolymer;

[0037] S3: Blend 13.5 g of dipeptide-15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain a dipeptide-15-sodium alginate aqueous solution;

[0038] Add 10 g of functionalized copolymer, 0.03 g of oligopeptide-4, and 100 mL of tetrahydrofuran into a reaction kettle for ultrasonic emulsification, add 40 mL of dipeptide-15-sodium alginate aqueous solution into the reaction kettle and disperse evenly, remove tetrahydrofuran, concentrate to obtain a drug-loaded micelle with a solid content of 15%;

[0039] S4: Blend 0.5 g of α-lipoic acid and 100 mL of absolute ethanol, add 100 g of drug-loaded micelle, control the temperature at 40 °C, keep the temperature for 3 - 6 h under stirring conditions, ultrafilter and concentrate to obtain an active peptide microcapsule.

[0040] The preparation method of the active peptide microcapsule in Example 2 includes the following steps:

[0041] S1: Under nitrogen protection, add 100 g of monomethoxypolyethylene glycol (MW = 2000), 250 g of ε-caprolactone, and 0.2 g of stannous octoate into a reaction kettle, disperse them evenly, control the temperature at 75 °C, keep the temperature for reaction for 24 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0042] S2: After blending 100 g of copolymer, 10 g of histidine, 5 g of N-isopropylacrylamide, and 1500 mL of N,N-dimethylformamide, blend 15 g of EDC and 7 g of NHS and add them, control the temperature at 60 °C, keep the temperature for reaction for 12 h, dialyze to remove unreacted monomers, and dry to obtain a functionalized copolymer;

[0043] S3: Blend 13.5 g of dipeptide-15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain a dipeptide-15-sodium alginate aqueous solution;

[0044] Add 10 g of the functional copolymer, 0.03 g of oligopeptide-4, and 150 mL of tetrahydrofuran to a reaction kettle for ultrasonic emulsification. Add 40 mL of the aqueous solution of dipeptide-15-sodium alginate to the reaction kettle and disperse evenly. Remove the tetrahydrofuran, concentrate, and obtain drug-loaded micelles with a solid content of 15%.

[0045] S4: Blend 1.2 g of α-lipoic acid and 150 mL of absolute ethanol, add 100 g of the drug-loaded micelles, control the temperature at 45 °C, keep warm under stirring conditions for 4.5 h, ultrafilter and concentrate to obtain active peptide microcapsules.

[0046] The preparation method of the active peptide microcapsules in Example 3 includes the following steps:

[0047] S1: Under nitrogen protection, add 100 g of monomethoxypolyethylene glycol (MW = 2000), 300 g of ε-caprolactone, and 0.25 g of stannous octoate to a reaction kettle and disperse evenly. Control the temperature at 80 °C, keep warm and react for 36 h, add cold ethyl ether for precipitation purification and drying to obtain the copolymer.

[0048] S2: Blend 100 g of the copolymer, 12 g of histidine, 6 g of N-isopropylacrylamide, and 2000 mL of N,N-dimethylformamide, then add the blend of 20 g of EDC and 5 - 10 g of NHS, control the temperature at 65 °C, keep warm and react for 16 h, dialyze to remove unreacted monomers, and dry to obtain the functional copolymer.

[0049] S3: Blend 13.5 g of dipeptide-15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain an aqueous solution of dipeptide-15-sodium alginate.

[0050] Add 10 g of the functional copolymer, 0.03 g of oligopeptide-4, and 200 mL of tetrahydrofuran to a reaction kettle for ultrasonic emulsification. Add 40 mL of the aqueous solution of dipeptide-15-sodium alginate to the reaction kettle and disperse evenly. Remove the tetrahydrofuran, concentrate, and obtain drug-loaded micelles with a solid content of 15%.

[0051] S4: Blend 2 g of α-lipoic acid and 200 mL of absolute ethanol, add 100 g of the drug-loaded micelles, control the temperature at 50 °C, keep warm under stirring conditions for 6 h, ultrafilter and concentrate to obtain active peptide microcapsules.

[0052] The preparation method of a peptide composition for brightening the skin in Example 4 includes the following steps:

[0053] Add 15.3 g of the active peptide microcapsules prepared in Example 1, 1.2 g of 1,3-propanediol, 0.225 g of caprylyl glycol, 0.075 g of ethylhexylglycerin, and 83.2 g of water to a preparation pot, start stirring for physical mixing to obtain the peptide composition.

[0054] Example 5 A preparation method of a peptide composition for brightening the skin, comprising the following steps:

[0055] Add 15.3 g of the active peptide microcapsules prepared in Example 2, 1.2 g of 1,3 - propanediol, 0.225 g of caprylyl glycol, 0.075 g of ethylhexylglycerin, and 83.2 g of water into a preparation pot, start stirring for physical mixing to obtain a peptide composition.

[0056] Example 6 A preparation method of a peptide composition for brightening the skin, comprising the following steps:

[0057] Add 15.3 g of the active peptide microcapsules prepared in Example 3, 1.2 g of 1,3 - propanediol, 0.225 g of caprylyl glycol, 0.075 g of ethylhexylglycerin, and 83.2 g of water into a preparation pot, start stirring for physical mixing to obtain a peptide composition.

[0058] Comparative Example 1 The preparation method of active peptide microcapsules comprises the following steps:

[0059] S1: Under nitrogen protection, add 100 g of monomethoxypolyethylene glycol (MW = 2000), 250 g of ε - caprolactone, and 0.2 g of stannous octoate into a reaction kettle and disperse evenly. Control the temperature at 75 °C, keep the temperature for reaction for 24 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0060] S2: After blending 100 g of the copolymer, 10 g of histidine, and 1500 mL of N,N - dimethylformamide, add 15 g of EDC and 7 g of NHS after blending. Control the temperature at 60 °C, keep the temperature for reaction for 12 h, dialyze to remove unreacted monomers, and dry to obtain a functionalized copolymer;

[0061] S3: Blend 13.5 g of dipeptide - 15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain an aqueous solution of dipeptide - 15 - sodium alginate;

[0062] Add 10 g of the functionalized copolymer, 0.03 g of oligopeptide - 4, and 150 mL of tetrahydrofuran into a reaction kettle for ultrasonic emulsification, add 40 mL of the aqueous solution of dipeptide - 15 - sodium alginate into the reaction kettle and disperse evenly, remove tetrahydrofuran, concentrate to obtain a drug - loaded micelle with a solid content of 15%;

[0063] S4: Blend 1.2 g of α - lipoic acid and 150 mL of absolute ethanol, add 100 g of the drug - loaded micelle, control the temperature at 45 °C, keep the temperature for 4.5 h under stirring conditions, ultrafilter and concentrate to obtain active peptide microcapsules.

[0064] Comparative Example 2 The preparation method of active peptide microcapsules comprises the following steps:

[0065] S1: Under nitrogen protection, add 100 g of monomethoxy polyethylene glycol (MW = 2000), 250 g of ε-caprolactone, and 0.2 g of stannous octoate into a reaction kettle and disperse evenly. Control the temperature at 75 °C, keep the temperature for reaction for 24 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0066] S2: Blend 13.5 g of dipeptide-15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain a dipeptide-15-sodium alginate aqueous solution;

[0067] Add 10 g of the copolymer, 0.03 g of oligopeptide-4, and 150 mL of tetrahydrofuran into a reaction kettle for ultrasonic emulsification. Add 40 mL of the dipeptide-15-sodium alginate aqueous solution into the reaction kettle and disperse evenly. Remove tetrahydrofuran, concentrate to obtain drug-loaded micelles with a solid content of 15%;

[0068] S3: Blend 1.2 g of α-lipoic acid and 150 mL of absolute ethanol, add 100 g of the drug-loaded micelles, control the temperature at 45 °C, keep the temperature for 4.5 h under stirring conditions, ultrafilter and concentrate to obtain active peptide microcapsules.

[0069] The preparation method of the active peptide microcapsules in Comparative Example 3 includes the following steps:

[0070] S1: Under nitrogen protection, add 100 g of monomethoxy polyethylene glycol (MW = 2000), 250 g of ε-caprolactone, and 0.2 g of stannous octoate into a reaction kettle and disperse evenly. Control the temperature at 75 °C, keep the temperature for reaction for 24 h, add cold ethyl ether for precipitation purification and drying to obtain a copolymer;

[0071] S2: Blend 100 g of the copolymer, 10 g of histidine, 5 g of N-isopropylacrylamide, and 1500 mL of N,N-dimethylformamide. After that, blend 15 g of EDC and 7 g of NHS and add them. Control the temperature at 60 °C, keep the temperature for reaction for 12 h, dialyze to remove unreacted monomers, and dry to obtain a functionalized copolymer;

[0072] S3: Blend 13.5 g of dipeptide-15, 1 g of sodium alginate, and 85.5 g of deionized water to obtain a dipeptide-15-sodium alginate aqueous solution;

[0073] Add 10 g of the functionalized copolymer, 0.03 g of oligopeptide-4, and 150 mL of tetrahydrofuran into a reaction kettle for ultrasonic emulsification. Add 40 mL of the dipeptide-15-sodium alginate aqueous solution into the reaction kettle and disperse evenly. Remove tetrahydrofuran, concentrate to obtain active peptide microcapsules.

[0074] The preparation method of the active peptide microcapsules in Comparative Example 4 includes the following steps:

[0075] S1: Under nitrogen protection, 100 g of monomethoxy polyethylene glycol (MW = 2000), 250 g of ε-caprolactone, and 0.2 g of stannous octoate were added to a reaction kettle and dispersed evenly. The temperature was controlled at 75 °C, and the reaction was carried out for 24 h under heat preservation. Then, cold ethyl ether was added for precipitation, purification, and drying to obtain a copolymer.

[0076] S2: After blending 100 g of the copolymer, 10 g of histidine, 5 g of N-isopropylacrylamide, and 1500 mL of N,N-dimethylformamide, 15 g of EDC and 7 g of NHS were blended and added. The temperature was controlled at 60 °C, and the reaction was carried out for 12 h under heat preservation. Unreacted monomers were removed by dialysis and then dried to obtain a functionalized copolymer.

[0077] S3: 13.5 g of dipeptide-15 and 85.5 g of deionized water were blended to obtain an aqueous solution of dipeptide-15.

[0078] 10 g of the functionalized copolymer, 0.03 g of oligopeptide-4, and 150 mL of tetrahydrofuran were added to a reaction kettle for ultrasonic emulsification. 40 mL of the aqueous solution of dipeptide-15 was added to the reaction kettle and dispersed evenly. Tetrahydrofuran was removed, and then it was concentrated to obtain drug-loaded micelles with a solid content of 15%.

[0079] S4: 1.2 g of α-lipoic acid and 150 mL of absolute ethanol were blended and added to 100 g of the drug-loaded micelles. The temperature was controlled at 45 °C, and the mixture was stirred and kept warm for 4.5 h. Then, it was ultrafiltered and concentrated to obtain active peptide microcapsules.

[0080] Compared with Example 5, in Comparative Example 5, only the active peptide microcapsules prepared in Example 2 added in Example 5 were replaced with the active peptide microcapsules prepared in Comparative Example 1 in equal amount, and the other components and preparation methods were exactly the same as those in Example 5.

[0081] Compared with Example 5, in Comparative Example 6, only the active peptide microcapsules prepared in Example 2 added in Example 5 were replaced with the active peptide microcapsules prepared in Comparative Example 2 in equal amount, and the other components and preparation methods were exactly the same as those in Example 5.

[0082] Compared with Example 5, in Comparative Example 7, only the active peptide microcapsules prepared in Example 2 added in Example 5 were replaced with the active peptide microcapsules prepared in Comparative Example 3 in equal amount, and the other components and preparation methods were exactly the same as those in Example 5.

[0083] Compared with Example 5, in Comparative Example 8, only the active peptide microcapsules prepared in Example 2 added in Example 5 were replaced with the active peptide microcapsules prepared in Comparative Example 4 in equal amount, and the other components and preparation methods were exactly the same as those in Example 5.

[0084] Performance detection

[0085] (1) Promoting cell proliferation activity test

[0086] a. Cell seeding: Take human skin fibroblasts and HaCaT cells in the logarithmic growth phase and seed them into 96-well plates at a density of 8×10 3 cells / well, and culture them in a CO2 incubator for 24 h;

[0087] b. Experimental grouping: Set up a blank group, a sample group, and a positive control group (100 ng / mL EGF), with 3 parallels in each group;

[0088] c. Sample preparation: Blend oligopeptide-4 and dipeptide-15 with a mass ratio of 0.025:5 as a compound sample; Set five concentration gradients of 1%, 2%, 3%, 4%, and 5% of the compound sample in the cell proliferation experiment, and set two concentrations of 1% and 5% in the test of promoting cell ATP and antioxidant activity;

[0089] d. Sample addition and incubation: After culturing for 24 h, aspirate the culture medium, rinse once with 200 μL PBS, add 100 μL of serum-free medium containing the sample to the sample group, add 100 μL of serum-free medium to the blank wells, and the zero group has no cell seeding and only 100 μL of serum-free medium is added;

[0090] e. Detection: Observe the cell morphology in the 96-well plate under a microscope, add 10 μL of CCK8 reagent to the blank group, the experimental group, and the positive control group, set the blank wells and zero wells according to the instructions, and incubate in the CO2 incubator in the dark for 1 h. Measure the absorbance at λ = 450 nm using an enzyme-linked immunosorbent assay reader. The calculation of cell viability is shown in the following formula:

[0091] U = (A1 - A0) / (A2 - A0):

[0092] In the formula, U - cell viability; A0 - absorbance of the zero well; A1 - absorbance of the test well; A2 - absorbance of the blank well; The calculation results are shown in Table 1;

[0093] Table 1: Statistical table of test results for promoting cell proliferation activity

[0094]

[0095] As can be seen from Table 1, the compounded oligopeptide-4 and dipeptide-15 in this application have a synergistic effect and significantly promote the proliferation of human skin fibroblasts.

[0096] (2) Test for promoting cell ATP production

[0097] a. Cell seeding: Take HaCaT cells in the logarithmic growth phase within 10 passages and seed them into 6-well plates at a density of 4×10 5 cells / well, and incubate them in a CO2 incubator for 24 h;

[0098] b. Experimental grouping: The experiment set up a blank group, a negative control group, a positive control (ergothioneine at 100 μg / mL), and a sample group, with 3 replicates in each group;

[0099] c. Sample incubation: After culturing for 24 h, discard the culture medium. After rinsing once with 1 mL of PBS, add 2 mL of serum-free medium containing the sample to the sample group, add 2 mL of serum-free medium containing ergothioneine to the positive control group, and add 2 mL of serum-free medium to the blank wells and negative control group and continue to incubate for 24 h;

[0100] d. Establishment of oxidative damage model: After incubating the sample with the cells for 24 h, discard the culture medium, and add 400 μM H2O2 to stimulate for 6 h except for the blank group;

[0101] e. Collection and detection of samples: After stimulation with H2O2, aspirate and discard the supernatant, carefully rinse once with 1 mL of PBS, and add 200 μL of ATP lysis solution in the ATP detection kit to each well to lyse the cells on ice;

[0102] f. BCA protein quantification: Take 20 μL of cell lysate and add it to a 96-well plate, then add 200 μL of BCA chromogenic solution and incubate at 37 °C for 30 min. Measure the OD value at a wavelength of 562 nm for protein correction.

[0103] g. ATP content detection: Operate and calculate according to the instructions in the enhanced ATP detection kit. The calculation results are shown in Table 2;

[0104] Table 2: Statistical table of the detection results of promoting cell ATP production

[0105]

[0106] As can be seen from Table 2, the compounded oligopeptide-4 and dipeptide-15 in this application have a synergistic effect, significantly promoting the release of cell ATP and having the effect of enhancing mitochondrial function.

[0107] (3) Antioxidant activity test

[0108] a. Cell seeding: Take HaCaT cells in the logarithmic growth phase and seed them in a 96-well plate with a black edge and a clear bottom at a density of 1.5×10 4 / well, and incubate in a CO2 incubator for 24 h;

[0109] b. Sample incubation: Discard the culture medium in each well of the well plate. Add DMEM basal medium containing a certain concentration of the test substance to the test substance wells and the positive control group, and add DMEM basal medium to the negative control, blank control, and naked cell wells. The liquid added to each well is 100 μL. After dosing, incubate in a CO2 incubator for 24 h;

[0110] c. DCFH-DA fluorescence incubation: Except for the naked cells, discard the culture medium in the remaining wells. After washing twice with PBS, add 100 μL of DCFH-DA (10 μM) working solution to each well and incubate in a CO2 incubator for 25 min;

[0111] d. H2O2 stimulation: Wash the cells in each well twice with PBS. Induce with H2O2 for 25 min for all wells except the blank control and the naked cells;

[0112] e. After the incubation is completed, wash the cells in each well twice with PBS and add 100 μL of PBS;

[0113] f. Place it in a fluorescence microplate reader, set the incident wavelength to 525 nm and the excitation wavelength to 488 nm, and read the value;

[0114] g. Calculate the relative fluorescence intensity of ROS of the test substance according to the following formula

[0115] Relative fluorescence intensity of ROS = [T - C0] / (C - C0)

[0116] In the formula: T - fluorescence intensity of the experimental group or negative control group; C - fluorescence intensity of the negative control; C0 - fluorescence intensity of the naked cell group; The calculation results are shown in Table 3;

[0117] Table 3: Statistical table of antioxidant activity detection results

[0118]

[0119] As can be seen from Table 3, the compounded oligopeptide-4 and dipeptide-15 in this application act synergistically to significantly inhibit the production of cellular ROS, achieving the effect of inhibiting oxidative stress.

[0120] Please refer to Figure 1 , at the same concentration, the ROS scavenging ability of the compounded sample group containing oligopeptide-4 and dipeptide-15 is better than that of ergothioneine. It has excellent ROS scavenging ability and can effectively inhibit cellular oxidative stress.

[0121] (4) Test for promoting collagen regeneration

[0122] a. Light source: One 40 W UVA lamp tube with a wavelength of 350 - 360 nm and a peak value of 365 nm

[0123] b. After 40 BALB / c Nude nude mice are adaptively fed for 1 - 2 weeks, randomly divide them into 4 groups:

[0124] ① Blank control group: Without ultraviolet irradiation;

[0125] ② Negative control group: Ultraviolet irradiation dose of 240 mJ / cm 2 ; Irradiate 3 times a week for 2 consecutive weeks;

[0126] ③Positive control group: Apply a cream containing 10% VE on the skin surface of mice + ultraviolet irradiation dose of 240 mJ / cm 2 ; Irradiate 3 times a week for 2 consecutive weeks;

[0127] ④Oligopeptide-4 + dipeptide-15: Add the peptide composition prepared in Example 5 to the cream to prepare a cream containing 50 ppm oligopeptide-4 + dipeptide-15, and apply the above cream on the skin surface of mice + ultraviolet irradiation dose of 240 mJ / cm 2 ; Irradiate 3 times a week for 2 consecutive weeks;

[0128] c. Post-treatment: Decapitate the nude mice, take two pieces of skin of the same size at symmetric positions on the left and right sides of the nude mice, attach the dermis layer of the skin downward to the pre-prepared filter paper, immerse it in 4% paraformaldehyde solution for fixation for 24 h, perform dehydration, clearing and wax infiltration, and then perform embedding, sectioning, staining and microscopic examination;

[0129] Please refer to Figure 2 , Under UVA stimulation, the collagen fibers in the dermis layer of nude mice are broken. The compound sample containing oligopeptide-4 and dipeptide-15 can improve the collagen state, increase the content of collagen fibers, and arrange them orderly. It can promote the neogenesis of extracellular matrix at the animal level and exert the anti-photoaging effect.

[0130] (5) Transdermal efficiency:

[0131] a. Franz diffusion cell method: Use ex vivo pig skin or artificial skin membrane, and the receptor fluid is pH 7.4 PBS + 0.5% Tween 80 (simulated body fluid);

[0132] b. Fluorescent labeling: Couple dipeptide-15 with FITC (fluorescein isothiocyanate), label oligopeptide-4 with Cy5.5, and observe the transdermal path and micelle disassembly through a confocal microscope;

[0133] c. Transdermal efficiency: Sampling at 24 h, and HPLC is used to measure the cumulative permeation amount of dipeptide-15 and oligopeptide-4;

[0134] d. Activity retention rate: Compare the secondary structure (circular dichroism spectrum) and biological activity (such as fibroblast proliferation experiment) of the peptide before and after transdermal; The detection results are shown in Table 4:;

[0135] Table 4: Transdermal efficiency of the active peptide microcapsules prepared in Examples 1-3 and Comparative Examples 1-4

[0136]

[0137] As can be seen from Table 4, the active peptide microcapsules prepared in this application have high transdermal efficiency and activity retention rate. Adding the active peptide microcapsules prepared in this application to skin care products greatly improves the absorption efficiency of the active peptide.

[0138] (6) 40°C accelerated stability test

[0139] The active peptide microcapsules prepared in Examples 1-3 and Comparative Examples 1-4 were dispensed into light-proof vials and stored in an incubator at 40°C with a relative humidity of 75% ± 5%. After 30 days, samples were taken to detect the retention rates of dipeptide-15 and oligopeptide-4. The test results are shown in Table 5.

[0140] (7) Light stability test

[0141] a. Light source: xenon lamp (4500 ± 500 lx), maintained at 25°C, with UVA (320 - 400 nm) accounting for 10%.

[0142] b. Irradiate continuously for 7 days and take samples after 168 h.

[0143] c. Analysis of photodegradation products: Identify the degradation products by UPLC-QTOF-MS. The decline rate of the main component peak area ≤ 5%. Detect the degradation rates of dipeptide-15 and oligopeptide-4. The test results are shown in Table 5.

[0144] Table 5: Stability of the active peptide microcapsules prepared in Examples 1-3 and Comparative Examples 1-4

[0145]

[0146] As can be seen from Table 5, in this application, dipeptide-15 and oligopeptide-4 were processed to prepare active peptide microcapsules, effectively improving the storage stability of dipeptide-15 and oligopeptide-4.

[0147] The above has described in detail one embodiment of the present invention, but the content described is only the preferred embodiment of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made according to the scope of the present invention application should still fall within the scope covered by the patent of the present invention.

Claims

1. A method for preparing a peptide composition for brightening skin, characterized in that: The method comprises the following steps: mixing active peptide microcapsules, 1,3-propylene glycol, caprylyl glycol, ethylhexylglycerol and water to obtain a peptide composition; the active peptide microcapsules are loaded with oligopeptide-4 and dipeptide-15 as peptide active substances; The preparation method of the active peptide microcapsule comprises the following steps: S1: Under nitrogen protection, monomethoxy polyethylene glycol, ε-caprolactone and stannous octoate are added into a reaction kettle and dispersed evenly, the temperature is controlled at 70-80°C, the reaction is kept warm for 18-36 hours, cold ether is added for precipitation, purification and drying to obtain a copolymer; S2: After the copolymer, histidine, N-isopropylacrylamide and N,N-dimethylformamide are blended, EDC and NHS are blended and added, the temperature is controlled at 55-65°C, the reaction is kept warm for 8-16 hours, unreacted monomers are removed by dialysis, and the mixture is dried to obtain a functionalized copolymer; S3: adding the functionalized copolymer, oligopeptide-4 and tetrahydrofuran into a reaction kettle for ultrasonic emulsification, adding the dipeptide-15-sodium alginate aqueous solution into the reaction kettle for uniform dispersion, removing the tetrahydrofuran, concentrating and obtaining drug-loaded micelles with a solid content of 10-20%; S4: α-lipoic acid and anhydrous ethanol are mixed, and the drug-loaded micelles are added. The temperature is controlled at 40-50°C, and the mixture is kept warm for 3-6 hours under stirring conditions. The mixture is ultrafiltered and concentrated to obtain active peptide microcapsules.

2. The method for preparing a skin-lightening peptide composition according to claim 1, characterized in that: The oligopeptide-4 accounts for 0.01-0.05% of the total mass of the peptide composition; the 1,3-propylene glycol accounts for 1.0-1.5% of the total mass of the peptide composition; the caprylyl glycol accounts for 0.2-0.5% of the total mass of the peptide composition; and the ethylhexylglycerol accounts for 0.05-0.08% of the total mass of the peptide composition.

3. The method for preparing a skin-lightening peptide composition according to claim 2, characterized in that: The dipeptide-15 accounts for 3-8% of the total weight of the peptide composition.

4. The method for preparing a skin-lightening peptide composition according to claim 2, characterized in that: When dipeptide-15 accounts for 8-13% of the total mass of the peptide composition; the peptide composition further comprises butanediol; the butanediol accounts for 8-13% of the total mass of the peptide composition.

5. The method for preparing a skin-lightening peptide composition according to claim 1, characterized in that: The addition ratio of monomethoxy polyethylene glycol, ε-caprolactone and stannous octoate in S1 is 100g: 200-300g: 0.15-0.25g.

6. The method for preparing a skin-lightening peptide composition according to claim 1, characterized in that: The addition ratio of copolymer, histidine, N-isopropylacrylamide, N,N-dimethylformamide, EDC and NHS in S2 is 100g: 8-12g: 4-6g: 1000-2000mL: 10-20g: 5-10g.

7. The method for preparing a skin-lightening peptide composition according to claim 1, characterized in that: The dipeptide-15-sodium alginate aqueous solution in S3 includes 5-15wt% dipeptide-15 and 0.5-1wt% sodium alginate; The addition ratio of the functionalized copolymer, oligopeptide-4, tetrahydrofuran, and dipeptide-15-sodium alginate aqueous solution is 10 g: 0.01-0.1 g: 100-200 mL: 40 mL.

8. The method for preparing a skin-lightening peptide composition according to claim 1, characterized in that: The addition ratio of α-lipoic acid, anhydrous ethanol and drug-loaded micelles in S4 is 0.5-2g:100-200mL:100g.

9. A peptide composition for brightening skin, characterized in that: Prepared by the preparation method described in any one of claims 1 to 8.

10. The method for using the peptide composition according to claim 9, characterized in that: The peptide composition is used in skin care products, and the peptide composition accounts for 0.1-20 wt % of the quality of the skin care products.

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