Ascorbic acid polypeptide derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of organic compounds, in particular to an ascorbic acid polypeptide derivative and a preparation method and application thereof. The ascorbic acid polypeptide derivative provided by the invention comprises a product obtained by coupling ascorbic acid derivatives such as 3-O-ethyl ascorbic acid ether, 3-O-acetyl ascorbic acid ether, ascorbic acid glucoside, ascorbyl palmitate and the like with carnosine derivatives such as N-acetyl carnosine, N-palmitoyl carnosine, N-benzoyl carnosine and the like; the ascorbic acid polypeptide derivative structurally retains C = C double bonds in VC with high reducibility, meanwhile, a polypeptide structure is added, the problem that ascorbic acid is poor in stability can be well solved, and compared with single use of polypeptide, the anti-aging performance of the ascorbic acid polypeptide derivative is obviously improved; compared with single use of VC, the whitening effect is obviously improved.

Description

Technical Field

[0001] The present invention relates to the technical field of organic compounds, and in particular to ascorbic acid polypeptide derivatives and preparation methods and applications thereof. Background Art

[0002] Long-term exposure to ultraviolet rays can cause photoaging of the skin, which manifests as spots, wrinkles, sagging and uneven skin tone. These changes not only affect personal appearance, but may also cause more serious skin diseases. Therefore, the research and development of effective anti-photoaging skin care products has become an important topic in the current skin care field.

[0003] The core of anti-photoaging skin care products lies in the selection and combination of their active ingredients. Ideal skin care products should have the following characteristics: first, they can effectively absorb or reflect ultraviolet rays to reduce their direct damage to the skin; second, they contain antioxidants to neutralize free radicals generated by ultraviolet radiation and prevent cell damage; third, they can promote the repair and regeneration of skin cells and enhance the skin's own defense capabilities; fourth, they have a whitening effect, can reduce the production of melanin and brighten the skin tone.

[0004] L-Ascorbic Acid (Vitamin C) is a water-soluble substance. As a powerful antioxidant, it participates in the body's complex metabolic processes and has been widely used in the cosmetics industry. Its unique antioxidant, whitening and anti-aging functions make it a key ingredient in many skin care products. However, the enediol structure in the L-ascorbic acid molecule is extremely unstable in vitro, which makes vitamin C stable only in a water environment with a pH of <3.5. However, skin care products with a pH of <3.5 will cause strong stinging when applied directly to the skin, and can easily damage the skin barrier. In addition, when vitamin C is used in cosmetics at a high concentration, it causes greater irritation to the skin and may not be suitable for sensitive skin.

[0005] Therefore, stable and mild ascorbic acid derivatives can be more widely used in skin care products, and they can exert their effects after being enzymatically hydrolyzed into ascorbic acid in the skin. While maintaining the skin care effects of VC, such ascorbic acid derivatives improve the safety and convenience of their application.

[0006] Carnosine (L-Carnosine, β-alanyl-L-histidine) is a type of dimeric peptide molecule that exists in high concentrations in the brain and skeletal muscle tissue of vertebrates. Supplementation of carnosine is believed to help alleviate some age-related neurological diseases, such as Alzheimer's disease, Parkinson's disease and diabetes. Carnosine can capture free radicals and prevent cross-linking of glycosylation. It has good antioxidant and anti-glycation effects and is often added to skin care products as an anti-aging ingredient. However, the free amino group of carnosine is highly active, making the carnosine molecule unstable under physiological conditions. Therefore, the free amino group of carnosine is acylated to obtain a carnosine derivative, which not only has a more stable structure, but also has improved antioxidant and anti-glycation abilities compared to the prototype carnosine, and has a better transdermal effect.

[0007] Currently, the coupling sites of ascorbic acid peptide derivatives on the market are relatively simple, and almost all of them connect the 6-hydroxyl group of the L-ascorbic acid molecule to the peptide through an ester bond, and the structure is not novel.

[0008] Although there are many ascorbic acid derivatives, there are almost no reports on coupling ascorbic acid derivatives with carnosine derivatives. Therefore, coupling ascorbic acid derivatives with carnosine derivatives to obtain ascorbic acid polypeptide derivatives will show great application prospects in anti-oxidation, anti-glycation, anti-photoaging and whitening. Summary of the invention

[0009] In view of the above technical problems, the present invention proposes relevant technical solutions for ascorbic acid polypeptide derivatives and preparation methods and applications thereof.

[0010] The ascorbic acid polypeptide derivative provided by the present invention has a chemical structure as shown in any one of formulas (1) to (18); wherein formula (1) and formula (2) are products of coupling 3-O-ethyl ascorbic acid ether with N-acetylcarnosine; formula (3) and formula (4) are products of coupling 3-O-ethyl ascorbic acid ether with N-palmitoylcarnosine; formula (5) and formula (6) are products of coupling 3-O-ethyl ascorbic acid ether with N-benzoylcarnosine; formula (7) and formula (8) are products of coupling 3-O-acetyl ascorbic acid ether with N-acetylcarnosine; formula (9) and formula (10) are products of coupling 3-O-acetyl ascorbic acid ether with N-palmitoylcarnosine. Formula (11) and (12) are the coupling products of 3-O-acetyl ascorbyl ether and N-benzoyl carnosine; Formula (13) is the coupling product of ascorbyl glucoside and N-acetyl carnosine; Formula (14) is the coupling product of ascorbyl glucoside and N-palmitoyl carnosine; Formula (15) is the coupling product of ascorbyl glucoside and N-benzoyl carnosine; Formula (16) is the coupling product of ascorbyl palmitate and N-acetyl carnosine; Formula (17) is the coupling product of ascorbyl palmitate and N-palmitoyl carnosine; Formula (18) is the coupling product of ascorbyl palmitate and N-benzoyl carnosine; ; ; ; ; ; ; ; .

[0011] The method for preparing the ascorbic acid polypeptide derivative comprises the following steps: firstly activating the carboxyl group of raw material A, and then subjecting the raw material A to an esterification reaction with raw material B to obtain the ascorbic acid polypeptide derivative.

[0012] Raw material A is a carnosine derivative, as shown in any one of the structures of formula (19) to formula (21); wherein formula (19) is N-acetylcarnosine, formula (20) is N-palmitoylcarnosine, and formula (21) is N-benzoylcarnosine.

[0013] .

[0014] Raw material B is an ascorbic acid derivative, as shown in any one of the structures of formula (22) to formula (25); wherein formula (22) is 3-O-ethyl ascorbic acid ether, formula (23) is 3-O-acetyl ascorbic acid ether, formula (24) is ascorbic acid glucoside, and formula (25) is ascorbic acid palmitate.

[0015] .

[0016] The preparation method of the ascorbic acid polypeptide derivative comprises: reacting a carnosine derivative raw material A with a coupling reagent in a solvent under the catalysis of 4-dimethylaminopyridine; after the reaction is completed, filtering and removing the by-product (N,N'-dicyclohexylurea) to obtain a filtrate; then adding a raw material B of the ascorbic acid derivative to the filtrate to react with an acid binding agent, concentrating, drying, and freeze-drying to obtain a solid product, such as the ascorbic acid polypeptide derivative shown in formula (1) to formula (18).

[0017] The coupling agent used in the present invention includes at least one of DCC (N,N'-dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and CDI (N,N'-carbonyldiimidazole).

[0018] In the process of synthesizing the ascorbic acid polypeptide derivative of the present invention, the acid-binding agent serves to neutralize the acidic substances generated during the reaction to prevent them from having a negative impact on the reaction. The acid-binding agent may be an organic base and / or an inorganic base, such as at least one of pyridine, triethylamine, DIEA, sodium carbonate, potassium carbonate, and sodium acetate, but is not limited thereto.

[0019] In the process of synthesizing the ascorbic acid polypeptide derivative of the present invention, the organic solvent used can be at least one of dichloromethane, chloroform, DMF, and dichloroethane, but is not limited thereto.

[0020] The preparation method of the ascorbic acid polypeptide derivative comprises: using the Steglich esterification reaction, under the catalysis of DMAP (4-dimethylaminopyridine), reacting the carnosine derivative (raw material A) with a coupling reagent (such as N,N'-dicyclohexylcarbodiimide of DCC) at room temperature in an organic solvent (such as dichloromethane); after the reaction is completed, filtering to remove the by-product DCU (N,N'-dicyclohexylurea); then adding the ascorbic acid derivative (raw material B) and an acid-binding agent (such as triethylamine) to the filtrate for reaction, concentrating, drying, and freeze-drying; and obtaining the final product ascorbic acid polypeptide derivative represented by formula (1) to formula (18).

[0021] The preparation method of the above-mentioned ascorbic acid polypeptide derivative comprises: Step 1: Activation of carnosine derivatives: Dissolving a carnosine derivative (e.g., at least one of N-acetylcarnosine, N-palmitoylcarnosine, and N-benzoylcarnosine) in a solvent, adding a coupling reagent and 4-dimethylaminopyridine, and stirring, so that the free carboxyl group of the carnosine derivative first reacts with the coupling reagent to form an active ester, and then exchanges with 4-dimethylaminopyridine to form an active amide; after the reaction is completed, filtering to remove the byproduct DCU (N,N'-dicyclohexylurea), and obtaining a filtrate; Step 2: Synthesis of ascorbyl carnosine derivatives: Adding an ascorbic acid derivative (e.g., at least one of 3-O ethyl ascorbic acid ether, 3-O acetyl ascorbic acid ether, ascorbic acid glucoside, and ascorbic acid palmitate) and an acid-binding agent to the filtrate of step 1, stirring for reaction; after the reaction is completed, concentrating, drying, and freeze-drying to obtain a white powder ascorbic acid polypeptide derivative; The above preparation steps also include a separation and purification step three, which is specifically: the white powder obtained in step two is dissolved in dichloromethane, added into a silica gel column, and column chromatography is performed using an ethyl acetate / petroleum ether system to separate isomers.

[0022] The above-mentioned ascorbic acid polypeptide derivatives can be used in the preparation of cosmetics or medicines.

[0023] The ascorbic acid polypeptide derivatives can be used in the preparation of anti-aging and whitening cosmetics or medicines.

[0024] A cosmetic with the above-mentioned ascorbic acid polypeptide derivative as an active ingredient, wherein the concentration of the ascorbic acid polypeptide derivative in the cosmetic is 5 to 10000 ppm. The concentration can also be adjusted to 10 to 500 ppm, 10 to 200 ppm, 10 to 100 ppm, etc. according to the efficacy requirements.

[0025] The invention couples multiple sites of L-ascorbic acid derivatives with carnosine derivatives respectively to obtain 18 different ascorbic acid polypeptide derivatives, which have diverse molecular structures and functions.

[0026] The present invention develops a variety of ascorbic acid polypeptide derivatives, which have the dual effects of VC whitening and carnosine anti-aging, and have a synergistic effect.

[0027] The ascorbic acid polypeptide derivative of the present invention achieves the purpose of anti-aging through anti-oxidation, anti-glycation and anti-photoaging effects; and achieves whitening by reducing melanin content and inhibiting tyrosinase activity.

[0028] In addition to synthesizing the structural formula, the present invention can also synthesize stereoisomers, mixtures of stereoisomers, or salts thereof.

[0029] The ascorbic acid polypeptide derivatives provided by the present invention can be used to prepare skin care products with anti-aging effects. Within the scope that does not impair the effects of the present invention, various ingredients commonly used in the field of cosmetics and quasi-drugs can be appropriately compounded as needed, such as powder components, moisturizers, emulsifiers, thickeners, metal ion masking agents, pigments, pH adjusters, skin nutrients, vitamins, preservatives, antioxidants, antioxidant adjuvants, fragrances, etc.

[0030] The ascorbic acid polypeptide derivative provided by the present invention can also be prepared in the form of freeze-dried powder, which can greatly improve the stability of the polypeptide during storage and use, and still maintain a high degree of activity during use to exert its maximum efficacy.

[0031] The ascorbic acid polypeptide derivative provided by the present invention retains the C=C double bond in the highly reduced VC in structure, and at the same time adds a polypeptide structure, which can well solve the problem of poor stability of ascorbic acid. Compared with the use of polypeptide alone, its anti-aging performance is significantly improved; compared with the use of VC alone, its whitening effect is significantly improved.

[0032] The present invention combines VC and VC derivatives commonly found in cosmetic raw materials with carnosine derivatives having antioxidant and anti-glycation effects, and performs improved modifications to achieve a synergistic effect. This group of novel ascorbic acid polypeptide derivatives has both anti-aging and whitening effects, exhibits low cytotoxicity in a variety of cell models, and can exert its effects at the ppm level, with low irritation, and has broad application prospects in the fields of cosmetic skin care such as anti-wrinkle and whitening.

[0033] Compared with 3-O-ethyl ascorbyl acetylcarnosine synthesized by other methods, the 3-O-ethyl ascorbyl acetylcarnosine, 3-O-ethyl ascorbyl palmitoylcarnosine and 3-O-ethyl ascorbyl benzoylcarnosine provided by the present invention have higher yield and simpler preparation process; and have higher melanin production inhibition rate and tyrosinase activity inhibition rate on melanocytes. The ascorbic acid polypeptide derivatives prepared by the present invention have better economy and better efficacy.

[0034] By conducting safety and efficacy verification on human melanocytes, human keratinocytes and human fibroblast models, it was found that the ascorbic acid polypeptide derivative provided by the present invention can obtain significant anti-aging and whitening effects at a concentration of 10-200 ppm, including inhibiting the generation of reactive oxygen species ROS, reducing melanin content, inhibiting tyrosinase activity, reducing the content of glycation end products, etc.; indicating that the ascorbic acid polypeptide derivative with a new structure prepared by the present invention can be used in cosmetics under the condition of meeting economic conditions.

[0035] The anti-aging essence provided by the present invention containing 3-O-ethyl ascorbyl acetylcarnosine, 3-O-ethyl ascorbyl palmitoylcarnosine and 3-O-ethyl ascorbyl benzoylcarnosine, after being used by subjects for 14 days and 28 days, significantly improves the water content of the stratum corneum of the subjects' skin, the glossiness and the elasticity of the skin; the average volume and area ratio of cheek wrinkles are significantly reduced, and the redness of the skin is also significantly improved, which has excellent anti-aging and repair effects.

[0036] The raw material components provided by the invention are green and safe, have low cytotoxicity, and have excellent effects, thus meeting the needs of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : H NMR spectrum of 3-O-ethyl ascorbyl acetylcarnosine.

[0038] Figure 2 : Carbon NMR spectrum of 3-O-ethyl ascorbyl acetylcarnosine.

[0039] Figure 3 : HPLC spectrum of 3-O-ethyl ascorbyl acetylcarnosine.

[0040] Figure 4:Effect of 3-O-ethyl ascorbyl acetylcarnosine on the cell survival rate of human primary keratinocytes and human primary melanocytes at different concentrations.

[0041] Figure 5 : The inhibitory effect of each group of samples in Example 3 on melanin content.

[0042] Figure 6 : The inhibitory effect of each group of samples in Example 3 on tyrosinase activity.

[0043] Figure 7 : The inhibitory effect of each group of samples in Example 4 on ROS.

[0044] Figure 8 : Fluorescence photos of cellular ROS content in each group of samples in Example 4.

[0045] Fig. 9 : The detection results of the anti-glycation effect of each group of samples in Example 5.

[0046] Fig.10 : The stability of each group of samples in Example 6 at 43°C in the dark.

[0047] Fig.11 : The inhibitory effect of each group of samples in Example 7 on melanin content.

[0048] Fig.12 : The inhibitory effect of each group of samples in Example 7 on tyrosinase activity. DETAILED DESCRIPTION

[0049] The present invention will be described below in conjunction with specific embodiments, and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments are used to illustrate the present invention, rather than to limit the present invention.

[0050] Example 1 Synthesis of ascorbic acid polypeptide derivatives Take 3-O-ethyl ascorbyl acetylcarnosine as an example: .

[0051] Step 1: Activation of N-acetylcarnosine 2.68 g N-acetylcarnosine (CAS: 56353-15-2, formula 19, 10 mmol) was dissolved in 100 mL dichloromethane, 2.06 g DCC (N, N'-dicyclohexylcarbodiimide, 10 mmol) and 1.22 g DMAP (4-dimethylaminopyridine, 10 mmol) were added, and stirred at room temperature for 10 h to allow the free carboxyl group of N-acetylcarnosine to react with DCC to form an active ester, and then exchange with DMAP to form an active amide. After the reaction was completed, the byproduct DCU (N, N'-dicyclohexylurea) was removed by filtration to obtain a filtrate.

[0052] Step 2: Synthesis of 3-O-ethyl ascorbyl acetylcarnosine Add 2.04 g 3-O-ethyl ascorbic acid ether (CAS: 86404-04-8, formula 22, 10 mmol) and 2.02 g acid-binding agent triethylamine (20 mmol) to the filtrate of step 1 and stir at room temperature for 2 h. After the reaction is completed, the product is concentrated under reduced pressure using a rotary evaporator and the remaining product is dissolved in 10 mL ethyl acetate. Then, it is washed with 20 mL 5wt% Na2CO3 solution until the pH is 8.5; then, the mixture is allowed to stand for stratification, and the upper organic phase is pipetted into a flask with a pipette, and the ethyl acetate is concentrated using a rotary evaporator, and then lyophilized to obtain a white powder 3-O-ethyl ascorbic acid acetylcarnosine (about 4.1 g).

[0053] Step 3: Isolation and purification of ascorbic acid peptide derivatives The 3-O-ethyl ascorbyl acetyl carnosine powder obtained in step 2 is two isomers (C 19 H 26 N4O9, M=454.4). Dissolve 2 g of the white powder from step 2 in 2 mL of dichloromethane, then add it to a silica gel column and perform column chromatography using 1 L of ethyl acetate / petroleum ether=1:10 system. Allow the eluent to flow slowly through the column, collect the eluate, and separate two isomers. Use nuclear magnetic resonance spectroscopy for structural confirmation.

[0054] 1H NMR of formula (1): δ 1.27 (3H, t, J = 7.1 Hz), 1.86 (3H, s), 2.39 - 2.51 (2H, 2.45 (t, J = 6.5 Hz), 2.45 (t, J = 6.5 Hz)), 3.08 - 3.20 (2H, 3.14 (d, J = 6.7 Hz), 3.14 (d, J = 6.7 Hz)), 3.37 - 3.49 (2H, 3.43 (t, J = 6.5 Hz), 3.43 (t, J = 6.5 Hz)), 4.15 - 4.26 (2H, 4.21 (q, J = 7.1 Hz), 4.21 (q, J = 7.1 Hz)), 4.47 - 4.71 (4H, 4.53 (td, J = 7.1, 4.0 Hz), 4.62 (d, J = 7.1 Hz), 4.62 (d, J = 7.1 Hz), 4.65 (t, J = 6.7 Hz)), 5.14 (1H, d, J = 4.0 Hz), 6.94 (1H, d, J = 1.7 Hz), 7.69 (1H, d, J = 1.7 Hz); Total yield 29.8%.

[0055] 1H NMR of formula (2): δ 1.28 (3H, t, J = 7.1 Hz), 1.86 (3H, s), 2.39 - 2.51 (2H, 2.45 (t, J = 6.5 Hz), 2.45 (t, J = 6.5 Hz)), 3.09 - 3.21 (2H, 3.15 (d, J = 6.7 Hz), 3.15 (d, J = 6.7 Hz)), 3.37 - 3.59 (4H, 3.43 (t, J = 6.5 Hz), 3.43 (t, J = 6.5 Hz), 3.53 (d, J = 5.9 Hz), 3.53 (d, J = 5.9 Hz)), 4.16 - 4.27 (2H, 4.22 (q, J = 7.1 Hz), 4.22 (q, J = 7.1 Hz)), 4.38 (1H, td, J = 5.9, 4.0 Hz), 4.61 (1H, t, J = 6.7 Hz), 5.14 (1H, d, J = 4.0 Hz), 6.94 (1H, d, J = 1.7 Hz), 7.69 (1H, d, J = 1.7 Hz); Total yield 19.1%.

[0056] The structure of 3-O-ethyl ascorbyl acetylcarnosine (Formula 1) is confirmed as follows: (I) Nuclear magnetic resonance spectroscopy (NMR) Instrument: BRUKER AVANCE III 600 NMR instrument; Solvent: D2O (deuterated water); Spectrum: Nuclear magnetic resonance hydrogen spectrum (1H NMR, such as Figure 1 As shown), nuclear magnetic resonance carbon spectrum (13C NMR, as Figure 2 as shown).

[0057] (II) High-resolution mass spectrometry (MS) Instrument model: Thermo Fisher Q Exactive; Ion source: ESI source; Mobile phase: 100% aqueous solution containing 10% HCOOH; Test data: Molecular formula is C 19 H 26 N4O9, [M+H] + The theoretical value is 455.1773 and the measured value is 455.1150.

[0058] (III) HPLC Instrument model: SHIMADZU Prominence LC-20A high performance liquid chromatograph; Column: ACE Excel 5 C18-Amide, 250×4.6mm; Elution conditions: 5%-30% MeCN / H2O gradient elution for 25 minutes, peak time t=8.3 min.

[0059] Sample concentration: 1 mg / mL; Injection volume: 50 μL; Detection wavelength: 244 nm; Sample purity: >97%; Test results: The peaks are shown in Table 1 below, and the HPLC spectrum is as follows Figure 3 shown.

[0060] Table 1: HPLC peak conditions

[0061] Synthesis of formula (3) to formula (18): the carnosine derivative in step 1 is one of N-acetylcarnosine, N-palmitoylcarnosine or N-benzoylcarnosine, the ascorbic acid derivative in step 2 is one of 3-O-ethyl ascorbic acid ether, 3-O-acetyl ascorbic acid ether, ascorbic acid glucoside or ascorbic acid palmitate, and the other steps, reagents and separation and purification methods are consistent with the above-mentioned synthesis method.

[0062] Example 2 Cytotoxicity test of ascorbic acid polypeptide derivatives Experimental methods: (1) Cell seeding: Human primary keratinocytes or human primary melanocytes were cultured at a rate of 5×10 4 The cells were inoculated into 96-well plates at a density of 100 / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0063] (2) Experimental grouping: The experiment set up a zero adjustment group, a blank control group and a sample group. The sample group was set up with 7 concentration gradients, and 3 replicate wells were set up under each concentration gradient.

[0064] Table 2: Experimental groups

[0065] (3) Administration: Administration was performed when the cell plating rate in the 96-well plate reached 50%. 200 μL of culture medium was added to each well of the blank control group; no cells were inoculated in the zero adjustment group, and only 200 μL of cell culture medium was added. The sample group was prepared with serum-free culture medium and diluted to concentrations of 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, 0.003125%, and 0.0015625%, and 200 μL of culture medium containing samples of the corresponding concentration was added to each well; after administration, the 96-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 h.

[0066] (4) Detection: After the cells were incubated for 24 h, the CCK-8 method was used to detect the cell metabolic activity.

[0067] (5) Calculation of cell viability: Calculated according to the formula, cell viability (%) = (sample well OD - zero adjustment well OD) / (blank control well OD - zero adjustment well OD) × 100%.

[0068] Experimental results: CCK-8 test results Figure 4 As shown. Figure 4The experimental results show that 3-O-ethyl ascorbyl acetyl carnosine does not show obvious cytotoxicity in the concentration range of 0.01% based on human primary keratinocytes. 3-O-ethyl ascorbyl acetyl carnosine does not show obvious cytotoxicity in the concentration range of 0.1% based on human primary melanocytes. This shows that the prepared new ascorbic acid peptide derivative has low cytotoxicity and has broad application prospects in the field of skin care products.

[0069] Example 3 Whitening efficacy test of 3-O-ethyl ascorbyl acetylcarnosine Skin pigmentation is a common cosmetic problem that can be caused by a variety of factors, including ultraviolet radiation, hormonal changes, post-inflammatory pigmentation, and genetic factors. Melanin is the main pigment that determines skin color, and its production and distribution are regulated by complex physiological processes. Tyrosinase is a key enzyme in melanin synthesis, so inhibiting the activity of tyrosinase has become one of the mechanisms of action of many whitening agents.

[0070] Melanin content: Melanin is produced by melanocytes and is the most important determinant of skin color. By measuring the melanin content in melanocytes, the whitening effect of the sample can be reflected. The lower the melanin content, the better the whitening effect of the sample.

[0071] Tyrosinase activity: Tyrosinase is a key enzyme in the process of melanin synthesis, and its activity determines the amount of melanin formed. After the sample is treated, the higher the inhibition rate of tyrosinase activity in melanocytes, the less melanin is synthesized, indicating that the whitening effect of the sample is better.

[0072] Experimental methods: (1) Cell inoculation: Primary human melanocytes were cultured at a rate of 5×10 4 The cells were inoculated into 24-well plates at a density of 100 / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0073] (2) Experimental grouping: The experiment set up a blank control group, a UV irradiation group, a positive control group and a sample group. Three replicate wells were set for each group. The sample groups were 3-O-ethyl ascorbyl acetylcarnosine (group A), 3-O-ethyl ascorbyl ether (group B), N-acetylcarnosine (group C), and a physical mixture of 3-O-ethyl ascorbyl ether and N-acetylcarnosine (group D).

[0074] Table 3: Experimental design grouping

[0075] (3) UV irradiation: The UV irradiation group, positive control group and sample group were irradiated with UVA at a dose of 4.8 J / cm 2 .

[0076] (4) Dosing: According to the test scheme in Table 3, when the cell plating rate in the 24-well plate reaches 50%, the drug is administered in groups. The dosage per well is 1 mL. Each group has 3 replicate wells and incubated in an incubator (37 ℃, 5% CO2) for 24 h.

[0077] (5) Melanin content detection: At the end of the time action, the cells in each well were collected into a 1.5 mL centrifuge tube by digestion method. After centrifugation, the supernatant was discarded and resuspended with 1 mol / L NaOH containing 10% DMSO and placed in a water bath at 80°C for 2 h. After the water bath, 200 μL of liquid was drawn from each group and added to a 96-well plate. The absorbance OD405 at a wavelength of 405 nm was detected using a multifunctional microplate reader, and the melanin content inhibition rate was calculated according to the following formula.

[0078] Tyrosinase activity detection: At the end of the time action, each group of cells was washed twice with sterile DPBS, 400 μL of 1% Triton X-100 solution was added to each well, frozen at -80°C for 30 min, and then taken out and thawed at room temperature for 30 min. 100 μL of 0.1% L-DOPA was added to each well, incubated at 37°C for 2 h, and 200 μL was pipetted into a 96-well plate with a pipette. The OD475 value was read at a wavelength of 475 nm by an enzyme reader, and the tyrosinase inhibition rate of each group was calculated according to the following formula.

[0079] Inhibition rate (%) = (1-OD value of this group / OD value of UV irradiation group) × 100%.

[0080] Experimental results: All data are expressed as mean ± standard deviation, and the groups were compared using t-test. P < 0.05 is indicated as *, indicating a significant difference, and P < 0.01 is indicated as **, indicating an extremely significant difference. Figures 5-6 As shown, Figure 5 It is a bar graph showing the inhibitory effect of each group of samples on melanin content. Figure 6 It is a bar graph showing the inhibitory effect of each group of samples on tyrosinase activity.

[0081] Depend on Figures 5 and 6 It can be seen that ① compared with the BC group, the melanin content and tyrosinase activity of the NC group increased significantly, indicating that the UV irradiation stimulation was effective; compared with the NC group, the melanin content and tyrosinase activity of the PC group decreased significantly, indicating that the positive control of this test was effective.

[0082] ② Compared with the NC group, in the sample A group, acetyl carnosine 3-O-ethyl ascorbate at a concentration of 0.02%, in the sample B group, 3-O-ethyl ascorbic acid ether at a concentration of 0.01%, in the sample C group, N-acetyl carnosine at a concentration of 0.01%, and in the sample D group, a composition of 0.01% acetyl carnosine 3-O-ethyl ascorbate and 0.01% N-acetyl carnosine, based on human primary melanocytes, both the melanin content and tyrosinase activity decreased significantly. Among them, in the sample C group, 0.01 < p < 0.05, and both the melanin content and tyrosinase activity decreased significantly; in the sample A group, sample B group, and sample D group, p < 0.01, and both the melanin content and tyrosinase activity decreased extremely significantly, indicating that the sample groups have obvious effects on inhibiting melanin production and inhibiting tyrosinase activity.

[0083] ③ In the sample A group, acetyl carnosine 3-O-ethyl ascorbate at a low concentration of 0.02% had an inhibition rate of 21.5% on melanin and an inhibition rate of 30.9% on tyrosinase activity, which was significantly higher than that of the sample B group with 3-O-ethyl ascorbic acid ether (melanin inhibition rate of 16.8% and tyrosinase activity inhibition rate of 20.3%) and the sample C group with N-acetyl carnosine (melanin inhibition rate of 8% and tyrosinase activity inhibition rate of 12.4%). It was also higher than the simple mixture of 3-O-ethyl ascorbic acid ether and N-acetyl carnosine (melanin inhibition rate of 19.3% and tyrosinase activity inhibition rate of 27.0%), indicating that chemically modifying VC and carnosine to prepare conjugates has a synergistic effect in inhibiting melanin production and inhibiting tyrosinase activity, and the whitening effect is significantly better than that of the two raw material compounds and the physical mixture of the two raw material compounds. This result shows that chemically coupling VC derivatives and carnosine derivatives in the present invention is beneficial. The prepared ascorbic acid polypeptide derivatives not only improve the stability of VC but also do not lose the original whitening efficacy of VC, but instead enhance the whitening effect, which may be related to the improved structural stability of VC, the antioxidant and anti-glycation properties of carnosine, etc.

[0084] Example 4: Photoprotective efficacy test of palmitoyl carnosine ascorbate Oxidative stress refers to the imbalance state between free radicals and the antioxidant defense system in the body. Under ultraviolet irradiation, the body will generate a large number of free radicals. Free radicals are highly reactive molecules with unpaired electrons, which can trigger chain reactions, leading to lipid peroxidation of cell membranes, protein denaturation, and DNA damage, etc., thereby promoting the occurrence and development of various diseases, including cardiovascular diseases, neurodegenerative diseases, diabetes, and skin aging, etc. Some studies have shown that L-carnosine, as an antioxidant, can protect cells from oxidative stress by scavenging free radicals, inhibiting free radical generation, or repairing oxidative damage. Vitamin C, as a naturally occurring antioxidant, has attracted much attention due to its strong reducing ability and extensive biological functions.

[0085] Reactive oxygen is considered to be an important cause of skin aging, which can cause thinning of the skin and wrinkles. Ultraviolet radiation promotes the formation of intracellular reactive oxygen. By detecting the content of ROS in keratinocytes, the antioxidant effect of the sample can be reflected.

[0086] Experimental methods: (1) Cell seeding: Human primary keratinocytes were cultured at a rate of 5×10 4 The cells were inoculated into 24-well plates at a density of 100 / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0087] (2) Experimental grouping: The experiment set up a blank control group, a UV irradiation group, a positive control group and a sample group. Three replicate wells were set for each group. The sample groups were ascorbyl palmitate benzoylcarnosine (group A), ascorbyl palmitate (group B), N-benzoylcarnosine (group C), and a physical mixture of ascorbyl palmitate and N-benzoylcarnosine (group D).

[0088] Table 4: Experimental design grouping

[0089] (3) UV irradiation: The UV irradiation group, positive control group and sample group were irradiated with UVA at a dose of 4.8 J / cm 2 .

[0090] (4) Dosing: After irradiation, according to the test scheme in Table 4, when the cell plating rate in the 24-well plate reaches 50%, the drug is administered in groups. The dosage per well is 1 mL, and each group has 3 replicate wells. The cells are incubated in an incubator (37°C, 5% CO2) for 24 h.

[0091] (5) ROS content detection: Wash the cells in each well twice with serum-free culture medium, add 10 μmol / L ROS dye DCFH-DA, incubate at 37 °C in the dark for 20 min, and wash again with serum-free culture medium three times. Under a fluorescence microscope with a 20× objective lens, randomly select one field of view from each well to take a picture, with three replicates in each group. After taking the picture, use Image J software to calculate the average fluorescence intensity of ROS, and calculate the ROS inhibition rate according to the following formula.

[0092] ROS inhibition rate (%) = (1-average fluorescence intensity of this group / average fluorescence intensity of UV irradiation group) × 100%.

[0093] Experimental results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. t-test was used for statistical analysis for comparison among the groups. P < 0.05 was indicated as *, which was considered to be significantly different; P < 0.01 was indicated as **, which was considered to be extremely significantly different. Figure 7-8 As shown, Figure 7 It is a bar graph of the inhibitory effect of each group of samples on ROS. Figure 8 Fluorescence photos of ROS content in cells of each group.

[0094] Depend on Figure 7-8 It can be seen that ① compared with the BC group, the ROS content of the cells in the NC group increased significantly, indicating that the UV irradiation stimulation was effective; compared with the NC group, the ROS content of the cells in the PC group decreased significantly, indicating that the positive control of this test was effective.

[0095] ②Compared with the NC group, the ROS content of samples A group ascorbyl palmitate benzoyl carnosine at a concentration of 10 ppm, B group ascorbyl palmitate at a concentration of 5 ppm, C group N-benzoyl carnosine at a concentration of 5 ppm, and D group 5 ppm ascorbyl palmitate and 5 ppm N-benzoyl carnosine combined, based on human primary keratinocytes, was significantly reduced. This indicates that the four groups of samples can significantly remove a large amount of reactive oxygen species in skin cells after ultraviolet irradiation, and achieve the effect of anti-photoaging through anti-oxidation.

[0096] ③ At a low concentration of 10 ppm, the inhibition rate of ROS of ascorbyl palmitate benzoyl carnosine in sample group A was as high as 61.5%, which was comparable to the positive control 10 mg / mL VC ethyl ether (ROS inhibition rate 60.9%), higher than 5 ppm ascorbyl palmitate in sample group B (ROS inhibition rate 54.2%) and 5 ppm N-benzoyl carnosine in sample group C (ROS inhibition rate 53.0%), and also higher than the simple mixture of ascorbyl palmitate and N-benzoyl carnosine at the same concentration (ROS inhibition rate 58.6%), indicating that coupling VC palmitate with N-benzoyl carnosine can produce a powerful antioxidant effect, and has a synergistic effect in inhibiting ROS generated by UV irradiation, thereby achieving anti-photoaging and anti-aging effects, which is better than the two raw material compounds and the physical mixture of the two raw material compounds. Therefore, the ascorbyl palmitate benzoyl carnosine prepared by the present invention has the dual effects of N-benzoyl carnosine anti-oxidation and VC anti-oxidation and whitening, has low cytotoxicity, low effective concentration, and significant effect, showing a broad application prospect in skin care products.

[0097] Example 5 Anti-glycation efficacy test of ascorbyl glucoside palmitoyl carnosine Glycation refers to the non-enzymatic glycosylation reaction of proteins, that is, under non-enzymatic conditions, the free amino groups of macromolecules such as proteins, amino acids, lipids or nucleic acids and the carbonyl groups of reducing sugars undergo a series of reactions such as condensation, rearrangement, cleavage, and oxidation modification, and finally form advanced glycation end products (AGEs). AGEs are closely related to skin aging. Excessive AGEs can undergo glycation and cross-linking reactions with skin elastic fibers and collagen, and AGEs are brown, which eventually causes the skin to yellow and reduce elasticity. Carboxymethyl lysine (CML) is a structural form of AGEs. The less CML content, the more significant the anti-glycation effect of the sample.

[0098] Experimental methods: Human dermal fibroblasts were seeded in 12-well plates (5 × 10 4 / well), incubate in a 37 ℃, 5% CO2 incubator for 24 h. Remove the culture medium and gently rinse the cells once or twice with D-Hanks. The blank control group was replaced with fresh culture medium, the sample group was replaced with fresh culture medium containing modeling agent (MGO) and sample, the model control group was replaced with fresh culture medium containing modeling agent (MGO), and the positive control group was replaced with fresh culture medium containing modeling agent (MGO) and aminoguanidine, and continued to incubate in the incubator for 48 h. The experimental design grouping is shown in Table 5 below: Table 5: Experimental design grouping

[0099] After the incubation, the solution was discarded and washing-fixation-permeabilization-antibody incubation was performed. Photos were taken under a fluorescence microscope, and the immunofluorescence results were quantitatively analyzed using Image J software. The glycation inhibition rate was calculated according to the following formula.

[0100] Glycation inhibition rate (%) = (1-mean fluorescence intensity of this group / mean fluorescence intensity of model control group) × 100%.

[0101] Experimental results: GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. t-test was used for statistical analysis between groups. P < 0.05 was indicated as *, which was considered to be significantly different; P < 0.01 was indicated as **, which was considered to be extremely significantly different. Fig. 9 shown.

[0102] from Fig. 9 It can be seen that ① compared with the BC group, the average fluorescence intensity of CML cells in the NC group increased significantly, indicating that the MGO modeling was effective; compared with the NC group, the average fluorescence intensity of CML cells in the PC group decreased significantly, indicating that the positive control of this test was effective.

[0103] ② Compared with the NC group, the average fluorescence intensity of CML in Group A of ascorbyl glucoside palmitoyl carnosine at a concentration of 0.02%, Group B of ascorbyl glucoside at a concentration of 0.01%, Group C of N-palmitoyl carnosine at a concentration of 0.01%, and Group D of a mixture of 0.01% ascorbyl glucoside and 0.01% N-palmitoyl carnosine based on human dermal fibroblasts all decreased significantly. Among them, in Group B, 0.01 < p < 0.05, and the average fluorescence intensity of CML decreased significantly; in Group A, Group C, and Group D, p < 0.01, and the average fluorescence intensity of CML decreased extremely significantly, indicating that the sample groups had an obvious effect on inhibiting advanced glycation end products.

[0104] ③ At a low concentration of 0.02%, the inhibition rate of ascorbyl glucoside palmitoyl carnosine in Group A of the sample on glycosylation reached 43.9%, which was higher than that of Group B of the sample with 0.01% ascorbyl glucoside (glycosylation inhibition rate of 14.2%) and Group C of the sample with 0.01% N-palmitoyl carnosine (glycosylation inhibition rate of 34.5%), and was also higher than the simple mixture of ascorbyl glucoside and N-palmitoyl carnosine at the same concentration (glycosylation inhibition rate of 35.6%). This shows that coupling VC glucoside with N-palmitoyl carnosine can produce a powerful anti-glycation effect, with a synergistic effect, which is superior to the individual action of the two raw material compounds and the physical mixture of the two raw material compounds.

[0105] Therefore, the ascorbyl glucoside N-palmitoyl carnosine prepared by the present invention has both the dual effects of N-palmitoyl carnosine's anti-glycation, anti-oxidation and VC's anti-oxidation and whitening, can effectively delay skin aging, help whiten the skin, and improve the problem of uneven skin tone.

[0106] Example 6 Stability Experiment of Ascorbic Acid Polypeptide Derivatives Experimental method: Prepare aqueous solutions of 3-O-ethyl ascorbic acid acetyl carnosine (Formula 1), 3-O-acetyl ascorbic acid acetyl carnosine (Formula 7), ascorbyl glucoside acetyl carnosine (Formula 13), ascorbyl palmitate acetyl carnosine (Formula 16) and the prototype L-ascorbic acid at a concentration of 50 mg / L respectively, place them in 50 mL glass bottles, and store them in the dark at 43 °C for 10 days. At the time points of day 0, day 1, day 3, day 6, and day 10, take out 400 μL of the solution from each bottle and pour it into a quartz cuvette, and detect its absorbance in a UV-visible spectrophotometer. Each group is measured in parallel three times. The stability is evaluated by measuring the decrease in absorbance of each group of samples at their maximum absorption wavelength. The stability calculation formula is as follows: Stability (%) = Absorbance on the nth day / Absorbance on day 0 × 100%.

[0107] Experimental results: GraphPad Prism was used to plot the results, and the results were expressed as Mean ± SD. Fig.10 shown.

[0108] from Fig.10 It can be seen that ① the original ascorbic acid only has about 20% left after 1 day, and only 8% after 10 days. Its stability is very poor, so its application in cosmetics is very limited. The reason is that it contains an enol structure, which is more active in chemical reactions and is prone to electron transfer or oxidation. In addition, it contains more free -OH in its molecules, has higher activity, and is easy to react with other substances, especially under aerobic conditions, these hydroxyls are easily oxidized. The stability of ascorbic acid peptide derivatives within 10 days is improved compared with the original ascorbic acid. It is speculated that this is because the ascorbic acid peptide derivatives reduce the activity of the enol hydroxyl group in the ascorbic acid molecule by changing its chemical structure, thereby reducing its possibility of being oxidized in light, heat and air.

[0109] ② Among the four derivatives, 3-O-ethyl ascorbyl acetyl carnosine has the best stability, retaining more than 90% integrity after 10 days, followed by 3-O-acetyl ascorbyl acetyl carnosine (88% remaining after 10 days), and ascorbyl palmitate acetyl carnosine (74% remaining after 10 days). The stability of ascorbyl glucoside acetyl carnosine is lower than that of the other three derivatives, which may be due to its large number of free hydroxyl groups and the easier degradation of sugar rings. However, its remaining amount after 10 days (28%) is still higher than that of the original VC (8%), indicating that the conjugate of VC derivatives and carnosine derivatives improves the stability of VC, greatly expanding the application scope of ascorbic acid peptide derivatives in the fields of cosmetic formulations and pharmaceutical preparations.

[0110] Example 7 Comparison of whitening effects of ascorbic acid polypeptide derivatives Purpose: The 3-O-ethyl ascorbyl acetylcarnosine (Formula 1), 3-O-ethyl ascorbyl palmitoylcarnosine (Formula 3), and 3-O-ethyl ascorbyl benzoylcarnosine (Formula 5) provided by the present invention were compared with the 3-O-ethyl ascorbyl carnosine described in Chinese patent CN 115260170 B in a melanocyte model for whitening efficacy (same concentration), thereby comparing the differences in whitening efficacy of the combination of different carnosine derivatives with ascorbic acid ethyl ether.

[0111] Synthesis of 3-O-ethyl ascorbyl carnosine: Compared with Chinese patent CN 115260170 B, the present invention improves the synthesis process of 3-O-ethyl ascorbic acid carnosine, and the specific steps are as follows: .

[0112] Step 1: Synthesis of N-Boc-carnosine Mix 25 mL of water with 10 mL of ethyl acetate, add 4.52 g of carnosine (CAS: 305-84-0, formula 26, 20 mmol), 4 g of sodium hydroxide, 10.6 g of sodium carbonate, and 6.1 g of triethylamine, place in a 30°C water bath, slowly drop 11 g of di-tert-butyl dicarbonate, and react for 5 h after the addition is complete. Cool the reaction solution to 0°C, maintain this temperature and drop 2 mol / L hydrochloric acid to adjust the system pH to 2, extract with ethyl acetate (20 mL×4), and wash the organic layer with saturated brine (20 mL×2). After drying with 20 g of anhydrous sodium sulfate, evaporate the solvent under reduced pressure to obtain a colorless oil. Add 5 mL of petroleum ether and stir. After a white solid appears, add a large amount of petroleum ether and stir, and filter to obtain N-Boc-carnosine.

[0113] Step 2: Activation of N-Boc-Carnosine 3.26 g N-Boc-carnosine (10 mmol) obtained in step 1 was dissolved in 100 mL dichloromethane, 2.06 g DCC (N,N'-dicyclohexylcarbodiimide, 10 mmol) and 1.22 g DMAP (4-dimethylaminopyridine, 10 mmol) were added, and stirred at room temperature for 10 h. After the reaction was completed, the byproduct DCU (N,N'-dicyclohexylurea) was removed by filtration to obtain a filtrate.

[0114] Step 3: Synthesis of 3-O-ethylascorbic acid-N-Boc-carnosine Add 2.04 g of 3-O-ethyl ascorbic acid ether (Formula 22, 10 mmol) and 2.02 g of acid-binding agent triethylamine (20 mmol) to the filtrate of step 2 and stir at room temperature for 2 h. After the reaction is completed, use a rotary evaporator to reduce pressure and concentrate, and then put it into a vacuum drying oven to dry. The solid material is recrystallized with 20 mL of isopropanol at 50°C, filtered, and the solid left on the filter paper is filtered to obtain 3-O-ethyl ascorbic acid-N-Boc-carnosine.

[0115] Step 4: Synthesis of 3-O-ethyl ascorbyl carnosine Add a mixed solution of 18 mL of trifluoroacetic acid and 20 mL of dichloromethane to the entire product obtained in step 3, stir at room temperature for 1 h, and remove the N-Boc protecting group. After the reaction is stopped, the solvent is concentrated and removed by rotary evaporation, and the remaining product is dissolved in 10 mL of ethyl acetate. Then, wash with 20 mL of 5wt% Na2CO3 solution by stirring until the pH is 8.5; then stand and separate, draw the organic phase with a pipette and use a rotary evaporator to concentrate and remove the ethyl acetate, and then freeze-dry to obtain a white powder 3-O-ethyl ascorbic acid carnosine (Formula 27).

[0116] The structure of 3-O-ethyl ascorbic acid carnosine prepared by the above method is shown in formula (27), and its nuclear magnetic resonance hydrogen spectrum structure is identified as follows: Equation (27) 1H NMR: δ 1.27 (3H, t, J = 7.1 Hz), 2.36-2.47 (2H, 2.41 (t, J =6.6 Hz), 2.41 (t, J = 6.6 Hz)), 2.78-2.90 (2H, 2.84 (t, J = 6.6 Hz), 2.84 (t,J = 6.6 Hz)), 3.08-3.20 (2H, 3.14 (d, J = 6.7 Hz), 3.14 (d, J = 6.7 Hz)), 4.15-4.26 (2H, 4.21 (q, J = 7.1 Hz), 4.21 (q, J = 7.1 Hz)), 4.47-4.70 (4H,4.53 (td, J = 7.1, 4.0 Hz), 4.62 (d, J = 7.1 Hz), 4.62 (d, J = 7.1 Hz), 4.65(t, J = 6.7 Hz)), 5.14 (1H, d, J = 4.0 Hz), 6.94 (1H, d, J = 1.7 Hz), 7.69 (1H, d, J = 1.7 Hz); the total yield is 24.5%.

[0117] According to the preparation method provided by the present invention, the total yield of the prepared 3-O-ethyl ascorbyl carnosine (Formula 27) reaches 24.5%, which is much higher than the yield obtained by the preparation method in Chinese patent CN115260170B (the yield recorded in paragraph 154 of its specification is 4.9%).

[0118] In vitro whitening efficacy test: (1) Cell inoculation: Primary human melanocytes were cultured at a rate of 5×10 4 The cells were inoculated into 24-well plates at a density of 100 / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0119] (2) Experimental grouping: The experiment set up a blank control group, a UV irradiation group, a positive control group and a sample group. Each group was set up with 3 replicate wells.

[0120] Table 6: Experimental design grouping

[0121] (3) UV irradiation: The UV irradiation group, positive control group and sample group were irradiated with UVA at a dose of 4.8 J / cm 2 .

[0122] (4) Dosing: According to the test scheme in Table 6, when the cell plating rate in the 24-well plate reaches 50%, the drug is administered in groups. The dosage per well is 1 mL. Each group has 3 replicate wells and incubated in an incubator (37 ℃, 5% CO2) for 24 h.

[0123] (5) The method for detecting melanin content and tyrosinase activity is the same as that in Example 3.

[0124] Experimental results: All data are expressed as mean ± standard deviation, and the groups were compared using t-test. P < 0.05 is indicated as *, indicating a significant difference, and P < 0.01 is indicated as **, indicating an extremely significant difference. Figures 11-12 As shown, Fig.11 It is a bar graph showing the inhibitory effect of each group of samples on melanin content. Fig.12 It is a bar graph showing the inhibitory effect of each group of samples on tyrosinase activity.

[0125] Depend on Figures 11-12It can be seen that ① the inhibition rates of 3-O-ethyl ascorbyl acetylcarnosine in sample group A, 3-O-ethyl ascorbyl palmitoylcarnosine in sample group B, and 3-O-ethyl ascorbyl benzoylcarnosine in sample group C on melanin at a concentration of 0.02% were 21.5%, 19.6%, and 20.4%, respectively, which were all higher than 3-O-ethyl ascorbyl carnosine in sample group D (inhibition rate 13.5%); the inhibition rates of tyrosinase activity in sample groups A, B, and C at a concentration of 0.02% were 30.9%, 29.2%, and 30.1%, respectively, which were all higher than 3-O-ethyl ascorbyl carnosine in sample group D (inhibition rate 21.5%). The experimental results show that the ascorbic acid polypeptide derivatives prepared by the present invention are better than Chinese patent CN115260170 B in terms of both the synthesis process and the in vitro whitening effect. In particular, the 3-O-ethyl ascorbyl acetyl carnosine in sample group A is most similar in structure to the 3-O-ethyl ascorbyl carnosine in patent CN115260170 B, with only the structure of the carnosine derivative being different. However, the former is significantly better than the latter in inhibiting melanin and tyrosinase activity, indicating that structural modification of the free amino group of carnosine can retain its biological activity and improve its stability to a greater extent.

[0126] Example 8 Anti-aging effects of serum containing ascorbic acid polypeptide derivatives on human body (1) Preparation of anti-wrinkle essence: 4% 1,3-butylene glycol, 0.5% betaine, 0.02% sodium hyaluronate, 0.08% xanthan gum, 0.3% phenoxyethanol and deionized water (added to 100%) were stirred and mixed to prepare a base essence. The products synthesized in Example 1, 3-O-ethyl ascorbyl acetyl carnosine (Formula 1, 10 ppm), 3-O-ethyl ascorbyl palmitoyl carnosine (Formula 3, 10 ppm), and 3-O-ethyl ascorbyl benzoyl carnosine (Formula 5, 10 ppm) were added to the base essence as an anti-aging composition, as sample group A. 3-O-ethyl ascorbyl carnosine of Chinese patent CN 115260170 B synthesized in Example 7 was added to the base essence at a mass concentration of 30 ppm, as sample group B.

[0127] (2) Volunteer selection: The selection of subjects follows the medical and ethical standards for human testing. All subjects must be willing to participate in the test and sign an informed consent form before the test. 60 healthy female subjects aged 18 to 40 were recruited and randomly divided into 2 groups, 30 in each group.

[0128] (3) Usage of experimental samples: After cleansing their faces in the morning and evening, the two groups of subjects used the essence of sample group A and group B for skin care, respectively. 0.2 g of essence was used each time and gently massaged until absorbed. Other anti-wrinkle skin care products were stopped during the experiment.

[0129] (4) Before using the samples and 14 days and 28 days after using the samples, the subjects' facial images were collected using VISIA-CR, and the subjects' images were collected using VC20 Plus to analyze the cheek skin roughness SEr and skin smoothness SEsm. The probes of German CK instruments were used: Corneometer CM 825 to measure the moisture content of the cheek stratum corneum, Glossymeter CL200 to measure the skin gloss, and Colorimeter CL400 to measure the skin color. SPSS analysis software was used to compare the measured values ​​at different time points with the baseline values ​​before using the samples, and the Shapiro-Wilk Test was used to perform a significance test for the normal distribution of the data improvement value.

[0130] Before the test, the subjects washed their faces with the same cleansing product and kept them in a stabilization room with constant temperature and humidity (temperature: 21 ℃ ± 1℃, humidity: 50% ± 10%) for 20 minutes to keep the skin condition stable, and then the above-mentioned test was carried out.

[0131] (5) The results are shown in the following table: The rate of change after using the product = (data after use - data before use) / data before use × 100%.

[0132] Table 7: Results of skin parameters before and after using anti-wrinkle essence

[0133] According to the data in Table 7, after using the essence containing 3-O-ethyl ascorbyl acetylcarnosine, 3-O-ethyl ascorbyl palmitoylcarnosine and 3-O-ethyl ascorbyl benzoylcarnosine for 14 days and 28 days, the water content of the stratum corneum, skin gloss and elasticity of the skin were significantly improved; and the average volume and area of ​​cheek wrinkles decreased significantly, and the redness of the skin was also significantly improved. Moreover, the parameter improvement of each index of the skin of the subject using sample A is better than that of sample B disclosed in patent CN 115260170 B. The above results illustrate that the essence containing 3-O-ethyl ascorbyl acetylcarnosine, 3-O-ethyl ascorbyl palmitoylcarnosine and 3-O-ethyl ascorbyl benzoylcarnosine provided by the present invention has the effects of moisturizing and locking water, reducing skin wrinkles, improving skin gloss, improving skin elasticity and repairing skin redness, making the skin look more energetic and more youthful in appearance, with significant anti-aging and repairing effects.

[0134] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An ascorbic acid polypeptide derivative, characterized in that: The chemical structure of the ascorbic acid polypeptide derivative is shown in any one of Formula (1) to Formula (18); Among them, formula (1) and formula (2) are the products of coupling 3-O-ethyl ascorbic acid ether and N-acetylcarnosine; Formula (3) and Formula (4) are the products of coupling 3-O-ethyl ascorbic acid ether with N-palmitoyl carnosine; Formula (5) and Formula (6) are the products of coupling 3-O-ethyl ascorbic acid ether with N-benzoylcarnosine; Formula (7) and Formula (8) are the products of coupling 3-O-acetyl ascorbic acid ether with N-acetyl carnosine; Formula (9) and Formula (10) are the products of coupling 3-O-acetyl ascorbic acid ether with N-palmitoyl carnosine; Formula (11) and Formula (12) are products of coupling 3-O-acetyl ascorbic acid ether with N-benzoylcarnosine; Formula (13) is the product of the coupling of ascorbyl glucoside and N-acetylcarnosine; Formula (14) is the product of the coupling of ascorbyl glucoside and N-palmitoyl carnosine; Formula (15) is the product of the coupling of ascorbyl glucoside and N-benzoylcarnosine; Formula (16) is the product of the coupling of ascorbyl palmitate and N-acetylcarnosine; Formula (17) is the product of the coupling of ascorbyl palmitate and N-palmitoyl carnosine; Formula (18) is the product of coupling ascorbyl palmitate with N-benzoylcarnosine; ； ； ； ； ； ； ； 。 2. A method for preparing the ascorbic acid polypeptide derivative according to claim 1, characterized in that: The method comprises the following steps: firstly activating the carboxyl group of raw material A, and then subjecting the raw material A to an esterification reaction with raw material B to obtain the ascorbic acid polypeptide derivative; Wherein, the raw material A is a carnosine derivative, as shown in the structure of any one of formulas (19) to (21); wherein formula (19) is N-acetylcarnosine, formula (20) is N-palmitoylcarnosine, and formula (21) is N-benzoylcarnosine; ； The raw material B is an ascorbic acid derivative, as shown in any one of the structures of formula (22) to formula (25); wherein formula (22) is 3-O-ethyl ascorbic acid ether, formula (23) is 3-O-acetyl ascorbic acid ether, formula (24) is ascorbic acid glucoside, and formula (25) is ascorbic acid palmitate; 。 3. The preparation method according to claim 2, characterized in that: The preparation method comprises: reacting a raw material A of a carnosine derivative with a coupling reagent in a solvent under the catalysis of 4-dimethylaminopyridine; after the reaction is completed, filtering and removing the byproduct N,N'-dicyclohexylurea to obtain a filtrate; then adding a raw material B of an ascorbic acid derivative to the filtrate to react with an acid binding agent, concentrating, drying, and freeze-drying to obtain a solid product, which is an ascorbic acid polypeptide derivative as shown in formula (1) to formula (18).

4. The preparation method according to claim 3, characterized in that: Acid binding agents include organic bases and / or inorganic bases; And / or, the coupling agent includes at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and N,N'-carbonyldiimidazole.

5. The preparation method according to claim 3, characterized in that: The solvent is at least one of dichloromethane, chloroform, DMF and dichloroethane.

6. Use of the ascorbic acid polypeptide derivative according to claim 1 in the preparation of cosmetics or medicines.

7. Use of the ascorbic acid polypeptide derivative according to claim 1 in the preparation of anti-aging, whitening cosmetics or medicines.

8. A cosmetic using the ascorbic acid polypeptide derivative according to claim 1 as an active ingredient, characterized in that: The concentration of ascorbic acid polypeptide derivatives in cosmetics is 5 to 10,000 ppm.

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