Ascorbic acid polypeptide derivatives, preparation methods thereof and applications

Abstract

The present invention relates to the technical field of organic compounds, and particularly to ascorbic acid polypeptide derivatives, their preparation methods and applications. The ascorbic acid polypeptide derivatives provided by the present invention include products obtained by coupling ascorbic acid derivatives such as 3-O-ethyl ascorbic acid ether, 3-O-acetyl ascorbic acid ether, ascorbic acid glucoside, ascorbic acid palmitate, etc. with carnosine derivatives such as N-acetyl carnosine, N-palmitoyl carnosine, N-benzoyl carnosine, etc.; the ascorbic acid polypeptide derivatives retain the C═C double bond in highly reducing VC in terms of structure, and at the same time add a polypeptide structure, which can well solve the problem of poor stability of ascorbic acid. Compared with using polypeptides alone, its anti-aging performance is significantly improved; compared with using VC alone, its whitening effect is significantly improved.

Description

Technical Field

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

[0002] Long-term exposure to ultraviolet light can cause photoaging of the skin, manifested as problems such as age spots, wrinkles, sagging, and uneven skin tone. These changes not only affect personal appearance but may also lead to more serious skin diseases. Therefore, the research and development of effective anti-photoaging skin care products have 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. An ideal skin care product should have the following characteristics: one is to be able to effectively absorb or reflect ultraviolet light, reducing its direct damage to the skin; the second is to contain antioxidants that can neutralize free radicals generated by ultraviolet irradiation to prevent cell damage; the third is to promote the repair and regeneration of skin cells and enhance the skin's own defense ability; the fourth is to have a whitening effect, which can reduce the production of melanin and brighten the skin tone.

[0004] L-Ascorbic Acid (L-Ascorbic Acid, Vitamin C) is a water-soluble substance. As a powerful antioxidant, it participates in the complex metabolic processes of the body 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, making vitamin C stable only in an aqueous environment with a pH < 3.5. However, skin care products with a pH < 3.5 directly acting on the skin will cause strong stinging and are extremely likely to damage the skin barrier. In addition, when the use concentration of vitamin C in cosmetics is relatively high, 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 exert their effects after being enzymatically hydrolyzed into ascorbic acid in the skin. While maintaining the skin care efficacy of VC, these ascorbic acid derivatives improve the safety and convenience of their applications.

[0006] Carnosine (L-Carnosine, β-alanyl-L-histidine) is a class of dimer peptide molecules that exist in high concentrations in the brains and skeletal muscle tissues of vertebrates. Supplementing carnosine is considered helpful in alleviating some age-related neurological diseases, such as Alzheimer's disease, Parkinson's disease, and diabetes. Carnosine can capture free radicals and prevent the cross-linking of glycosylation. It has good antioxidant and anti-glycation effects and is often added as an anti-aging ingredient in skin care products. However, the free amino group of carnosine has high activity, making the carnosine molecule unstable under physiological conditions. Therefore, acylating the free amino group of carnosine to obtain carnosine derivatives not only makes the structure more stable, but also improves its antioxidant and anti-glycation abilities compared to the original carnosine, and has a better transdermal effect.

[0007] Currently, the coupling sites of ascorbic acid polypeptide derivatives on the market are relatively single. Almost all are connecting the hydroxyl group at the 6th position of the L-ascorbic acid molecule to the polypeptide 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 aspects such as antioxidant, 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, their preparation methods, and applications.

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

[0011] ;

[0012] ;

[0013] ;

[0014] ;

[0015] ;

[0016] ;

[0017] ;

[0018] 。

[0019] The preparation method of the above ascorbic acid polypeptide derivative comprises the following steps: first, carboxyl activation is carried out on raw material A, and then an esterification reaction is carried out with raw material B to obtain the ascorbic acid polypeptide derivative.

[0020] Raw material A is a carnosine derivative, as shown by any one of the structures of formula (19) to formula (21); wherein, formula (19) is N-acetyl carnosine, formula (20) is N-palmitoyl carnosine, and formula (21) is N-benzoyl carnosine.

[0021] 。

[0022] Raw material B is an ascorbic acid derivative, as shown by 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.

[0023] 。

[0024] The preparation method of the above ascorbic acid polypeptide derivative includes: 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 to remove the by-product (N,N'-dicyclohexylurea) to obtain a filtrate; then adding a raw material B of an ascorbic acid derivative and a deacidifying agent to the filtrate for reaction, concentrating, drying, and freeze-drying to obtain a solid product, namely the ascorbic acid polypeptide derivative shown by formula (1) to formula (18).

[0025] The coupling agents used in the present invention include at least one of DCC (N,N'-dicyclohexylcarbodiimide), DIC (N,N'-diisopropylcarbodiimide), EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), and CDI (N,N'-carbonyldiimidazole).

[0026] In the process of synthesizing the ascorbic acid polypeptide derivative of the present invention, the role of the acid-binding agent is to neutralize the acidic substances generated during the reaction to prevent them from having a negative impact on the reaction. It can 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.

[0027] In the process of synthesizing the ascorbic acid polypeptide derivative of the present invention, the organic solvents used can be at least one of dichloromethane, chloroform, DMF, and dichloroethane, but are not limited thereto.

[0028] The preparation method of the above-mentioned ascorbic acid polypeptide derivative includes: using the Steglich esterification reaction, catalyzed by DMAP (4-dimethylaminopyridine), reacting a carnosine derivative (raw material A) with a coupling reagent (such as N,N'-dicyclohexylcarbodiimide of DCC) in an organic solvent (such as dichloromethane) at room temperature; after the reaction, filtering to remove the by-product DCU (N,N'-dicyclohexylurea); then adding an ascorbic acid derivative (raw material B) and an acid-binding agent (such as triethylamine) to the filtrate for reaction, concentrating, drying, and freeze-drying; obtaining the final product, namely the ascorbic acid polypeptide derivative of the compounds shown in formulas (1) to (18).

[0029] The preparation method of the above-mentioned ascorbic acid polypeptide derivative includes:

[0030] Step 1: Activation of the carnosine derivative:

[0031] Dissolve a carnosine derivative (such as at least one of N-acetylcarnosine, N-palmitoylcarnosine, and N-benzoylcarnosine) in a solvent, add a coupling reagent and 4-dimethylaminopyridine, and stir to first react the free carboxyl group of the carnosine derivative with the coupling reagent to form an active ester, and then exchange with 4-dimethylaminopyridine to form an active amide; after the reaction, filter to remove the by-product DCU (N,N'-dicyclohexylurea) to obtain a filtrate;

[0032] Step 2: Synthesis of the ascorbic acid carnosine derivative:

[0033] Add an ascorbic acid derivative (such as at least one of 3-O-ethyl ascorbate, 3-O-acetyl ascorbate, ascorbyl glucoside, and ascorbyl palmitate) and an acid-binding agent to the filtrate of Step 1, and stir for reaction; after the reaction, concentrate, dry, and freeze-dry to obtain a white powder of the ascorbic acid polypeptide derivative;

[0034] The above preparation steps further include step three of separation and purification, specifically: the white powder obtained in step two is dissolved in dichloromethane, silica gel column is added, and column chromatography is carried out with ethyl acetate / petroleum ether system to separate isomers.

[0035] The above ascorbic acid polypeptide derivative can be used in the preparation of cosmetics or drugs.

[0036] The above ascorbic acid polypeptide derivative can be used in the preparation of anti-aging, whitening cosmetics or drugs.

[0037] A cosmetic containing the above ascorbic acid polypeptide derivative as an active ingredient, and the concentration of the ascorbic acid polypeptide derivative in the cosmetic is 5 - 10000 ppm. The concentration can also be adjusted to 10 - 500 ppm, 10 - 200 ppm, 10 - 100 ppm, etc. according to the efficacy requirements.

[0038] In the present invention, multiple sites of L-ascorbic acid derivatives are respectively coupled with carnosine derivatives to obtain 18 different ascorbic acid polypeptide derivatives, which have molecular structure diversity and efficacy diversity.

[0039] The present invention has developed a variety of ascorbic acid polypeptide derivatives, which have both the dual effects of VC whitening and carnosine anti-aging, and have a synergistic effect.

[0040] The ascorbic acid polypeptide derivative of the present invention achieves anti-aging through antioxidant, anti-glycation and anti-photoaging effects; and realizes whitening by reducing the melanin content and inhibiting the activity of tyrosinase.

[0041] In addition to synthesizing the shown structural formula, the present invention can also synthesize its stereoisomers, or a mixture of its stereoisomers, or its salts.

[0042] The ascorbic acid polypeptide derivative provided by the present invention can be used to prepare skin care products with anti-aging efficacy. Within the range that does not damage the effects of the present invention, various components commonly used in the fields of cosmetics and quasi-drugs can be appropriately compounded as needed, such as powder components, moisturizers, emulsifiers, thickeners, metal ion masking agents, pigments, pH regulators, skin nutrients, vitamins, preservatives, antioxidants, antioxidant aids, fragrances, etc.

[0043] 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.

[0044] The ascorbic acid polypeptide derivative provided by the present invention retains the C═C double bond in highly reduced VC in terms of 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.

[0045] The present invention combines VC and common VC derivatives in cosmetic raw materials with carnosine derivatives having antioxidant and anti-glycation effects, and conducts improvement and modification, playing a synergistic effect. This group of novel ascorbic acid polypeptide derivatives has dual effects of anti-aging and whitening, shows low cytotoxicity in a variety of cell models, and can exert its effects at the ppm level, with little irritation, and has broad application prospects in the field of anti-wrinkle and whitening cosmetics and skin care.

[0046] The 3-O-ethylascorbic acid acetyl carnosine, 3-O-ethylascorbic acid palmitoyl carnosine, and 3-O-ethylascorbic acid benzoyl carnosine provided by the present invention have higher yields and simpler preparation processes compared with 3-O-ethylascorbic acid carnosine synthesized by other methods; the inhibition rates of melanin production and tyrosinase activity on melanocytes are higher. The ascorbic acid polypeptide derivatives prepared by the present invention have better economy and superior efficacy.

[0047] Through safety and efficacy verification on human melanocyte, human keratinocyte, and human fibroblast models, it is obtained 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 the melanin content, inhibiting tyrosinase activity, reducing the content of advanced glycation end products, etc.; indicating that under the condition of meeting economic conditions, the ascorbic acid polypeptide derivative with a brand-new structure prepared by the present invention can be applied to cosmetics.

[0048] The anti-aging essence containing 3-O-ethylascorbic acid acetyl carnosine, 3-O-ethylascorbic acid palmitoyl carnosine, and 3-O-ethylascorbic acid benzoyl carnosine provided by the present invention significantly improves the water content, skin gloss, and elasticity of the stratum corneum of the skin of the subjects after 14 days and 28 days of use by the subjects; while the average volume and area ratio of cheek wrinkles significantly decrease, and skin flushing is also significantly improved, having excellent anti-aging and repair effects.

[0049] The raw material components provided by the present invention are green, safe, have low cytotoxicity, and excellent effects, meeting the needs of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 : 1H NMR spectrum of 3-O-ethylascorbic acid acetyl carnosine.

[0051] Figure 2: Carbon-13 NMR spectrum of 3-O-ethylascorbic acid acetyl carnosine.

[0052] Figure 3 : HPLC spectrum of 3-O-ethylascorbic acid acetyl carnosine.

[0053] Figure 4 : Cell viability of 3-O-ethylascorbic acid acetyl carnosine on primary human keratinocytes and primary human melanocytes at different concentrations.

[0054] Figure 5 : Inhibitory effect of each group of samples in Example 3 on melanin content.

[0055] Figure 6 : Inhibitory effect of each group of samples in Example 3 on tyrosinase activity.

[0056] Figure 7 : Inhibitory effect of each group of samples in Example 4 on ROS.

[0057] Figure 8 : Fluorescence photographs of cellular ROS content of each group of samples in Example 4.

[0058] Figure 9 : Detection results of the anti-glycation effect of each group of samples in Example 5.

[0059] Figure 10 : Stability of each group of samples in Example 6 under dark conditions at 43°C.

[0060] Figure 11 : Inhibitory effect of each group of samples in Example 7 on melanin content.

[0061] Figure 12 : Inhibitory effect of each group of samples in Example 7 on tyrosinase activity. Detailed implementation manners

[0062] The present invention will be described below in conjunction with the detailed implementation manners, and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these detailed implementation manners are used to illustrate the present invention rather than limit the present invention.

[0063] Example 1 Synthesis method of ascorbic acid polypeptide derivatives

[0064] Taking 3-O-ethylascorbic acid acetyl carnosine as an example:

[0065] .

[0066] Step 1: Activation of N-acetyl carnosine

[0067] Dissolve 2.68 g of N-acetyl carnosine (CAS: 56353-15-2, Formula 19, 10 mmol) in 100 mL of dichloromethane, add 2.06 g of DCC (N,N'-dicyclohexylcarbodiimide, 10 mmol) and 1.22 g of DMAP (4-dimethylaminopyridine, 10 mmol), stir at room temperature for 10 h to first react the free carboxyl group of N-acetyl carnosine with DCC to form an active ester, and then exchange with DMAP to form an active amide. After the reaction is completed, filter to remove the by-product DCU (N,N'-dicyclohexylurea) to obtain a filtrate.

[0068] Step 2: Synthesis of 3-O-ethylascorbic acid acetyl carnosine

[0069] Add 2.04 g of 3-O-ethylascorbic acid ether (CAS: 86404-04-8, Formula 22, 10 mmol) and 2.02 g of the acid-binding agent triethylamine (20 mmol) to the filtrate from Step 1, and stir at room temperature for 2 h. After the reaction is completed, concentrate under reduced pressure using a rotary evaporator, dissolve the remaining product in 10 mL of ethyl acetate. Then wash with 20 mL of 5 wt% Na2CO3 solution until the pH is 8.5; then let it stand for layering, suck the upper organic phase into a flask with a pipette, concentrate to remove ethyl acetate using a rotary evaporator, and then lyophilize to obtain white powder 3-O-ethylascorbic acid acetyl carnosine (about 4.1 g).

[0070] Step 3: Separation and purification of ascorbic acid polypeptide derivatives

[0071] The 3-O-ethylascorbic acid acetyl carnosine powder obtained in Step 2 is a mixture of two isomers (C 19 H 26 N4O9, M = 454.4) shown in Formulas (1) to (2). Dissolve 2 g of the white powder from Step 2 in 2 mL of dichloromethane, then add it to a silica gel column, perform column chromatography using a system of ethyl acetate / petroleum ether = 1:10, let the eluent flow slowly through the column, collect the eluate, separate the two isomers, and use nuclear magnetic resonance spectroscopy for structural confirmation.

[0072] 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%.

[0073] 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%.

[0074] The structural confirmation of 3 - O - ethyl ascorbyl acetyl carnosine (Formula 1) is as follows:

[0075] (I) Nuclear Magnetic Resonance Spectroscopy (NMR)

[0076] Instrument: BRUKER AVANCE III 600 Nuclear Magnetic Resonance Spectrometer;

[0077] Solvent: D2O (deuterated water);

[0078] Spectra: Proton Nuclear Magnetic Resonance Spectrum (1H NMR, as Figure 1 shown), Carbon - 13 Nuclear Magnetic Resonance Spectrum (13C NMR, as Figure 2 shown).

[0079] (II) High - Resolution Mass Spectrometry (MS)

[0080] Instrument model: Thermo Fisher Q Exactive;

[0081] Ion source: ESI source;

[0082] Mobile phase: 100% aqueous solution containing 10% HCOOH;

[0083] Test data: The molecular formula is C 19 H 26 N4O9, [M + H] + The theoretical value is 455.1773, and the measured value is 455.1150.

[0084] (III) HPLC

[0085] Instrument model: SHIMADZU Prominence LC - 20A High - Performance Liquid Chromatograph;

[0086] Chromatographic column: ACE Excel 5 C18 - Amide, 250×4.6 mm;

[0087] Elution conditions: 5% - 30% MeCN / H2O gradient elution for 25 minutes, retention time t = 8.3 min.

[0088] Sample concentration: 1 mg / mL;

[0089] Injection volume: 50 μL;

[0090] Detection wavelength: 244 nm;

[0091] Sample purity: >97%;

[0092] Test results: The peaks are shown in Table 1 below, and the HPLC spectrum is as Figure 3 shown.

[0093] Table 1: HPLC Peak Elution Conditions

[0094]

[0095] Synthesis of Formulas (3) to (18): The carnosine derivative in Step 1 is one of N-acetyl carnosine, N-palmitoyl carnosine, or N-benzoyl carnosine, and 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. All other steps, reagents, and separation and purification methods are the same as those in the above synthesis method.

[0096] Example 2: Cytotoxicity Test of Ascorbic Acid Polypeptide Derivatives

[0097] Experimental Method:

[0098] (1) Cell Seeding: Human primary keratinocytes or human primary melanocytes were seeded into a 96-well plate at a density of 5×10 4 cells / mL and incubated overnight in an incubator (37 °C, 5% CO2).

[0099] (2) Experimental Grouping: The test was set up with a zero control group, a blank control group, and a sample group. The sample group was set with 7 concentration gradients, and 3 replicate wells were set for each concentration gradient.

[0100] Table 2: Experimental Grouping

[0101]

[0102] (3) Drug Administration: When the cell confluence rate in the 96-well plate reached 50%, drug administration was carried out. 200 μL of culture medium was added to each well in the blank control group; no cells were seeded in the zero control group, and only 200 μL of cell culture medium was added. The sample group was prepared and diluted to concentrations of 0.1%, 0.05%, 0.025%, 0.0125%, 0.00625%, 0.003125%, and 0.0015625% with serum-free medium, and 200 μL of culture medium containing the corresponding concentration of the sample was added to each well; after drug administration, the 96-well plate was placed in an incubator (37 °C, 5% CO2) and cultured for 24 h.

[0103] (4) Detection: After the cells were incubated for 24 h, the CCK-8 method was used to detect the cell metabolic activity.

[0104] (5) Calculation of Cell Survival Rate: Calculated according to the formula, cell survival rate (%) = (OD of sample well - OD of zero control well) / (OD of blank control well - OD of zero control well) × 100%.

[0105] Experimental Results:

[0106] The CCK-8 test results are as follows Figure 4 shown. According to Figure 4 the experimental results, it is considered that 3-O-ethylascorbic acid acetyl carnosine does not show obvious cytotoxicity based on human primary keratinocytes within the concentration range of 0.01%. 3-O-ethylascorbic acid acetyl carnosine does not show obvious cytotoxicity based on human primary melanocytes within the concentration range of 0.1%. It shows that the prepared novel ascorbic acid polypeptide derivative has low cytotoxicity and has broad application prospects in the field of skin care products.

[0107] Example 3 Whitening efficacy test of 3-O-ethylascorbic acid acetyl carnosine

[0108] Skin pigmentation is a common cosmetic problem, which may be caused by various factors, including ultraviolet radiation, hormonal changes, post-inflammatory hyperpigmentation, and genetic factors, etc. Melanin is the main pigment that determines skin color, and its production and distribution are regulated by complex physiological processes. Tyrosinase is the key enzyme in melanin synthesis, so inhibiting the activity of tyrosinase becomes one of the mechanisms of action of many whitening agents.

[0109] 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 efficacy of the sample can be reflected. The lower the melanin content, the better the whitening effect of the sample.

[0110] Tyrosinase activity: Tyrosinase is the key enzyme in the process of melanin synthesis, and the level of its activity determines the amount of melanin formed. After the action of the sample, the higher the inhibition rate of tyrosinase activity in melanocytes, the less melanin is synthesized, indicating that the whitening efficacy of the sample is better.

[0111] Experimental method:

[0112] (1) Cell seeding: Human primary melanocytes were seeded into 24-well plates at a density of 5×10 4 cells / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0113] (2) Experimental grouping: The experiment was set up with a blank control group, a UV irradiation group, a positive control group, and a sample group. Each group was set with 3 replicate wells. The sample group was divided into 3-O-ethylascorbic acid acetyl carnosine (Group A), 3-O-ethylascorbic acid ether (Group B), N-acetyl carnosine (Group C), and a physical mixture of 3-O-ethylascorbic acid ether and N-acetyl carnosine (Group D).

[0114] Table 3: Experimental design grouping

[0115]

[0116] (3) UV irradiation: The UV irradiation group, positive control group, and sample group were subjected to UVA irradiation at a dose of 4.8 J / cm 2 .

[0117] (4) Drug addition: According to the test protocol in Table 3, when the cell seeding rate in the 24-well plate reached 50%, grouping and drug administration were carried out. The drug dosage per well was 1 mL, and 3 replicate wells were set for each group. Incubate in an incubator (37 °C, 5% CO2) for 24 h.

[0118] (5) Melanin content detection: At the end of the time of action, the cells in each well were collected into 1.5 mL centrifuge tubes by digestion method. After centrifugation, the supernatant was discarded, and the cells were resuspended with 1 mol / L NaOH containing 10% DMSO and incubated in a water bath at 80 °C for 2 h. After the water bath, 200 μL of the liquid was taken from each group and added to a 96-well plate, and the absorbance OD405 at a wavelength of 405 nm was measured with a multifunctional microplate reader, and the inhibition rate of melanin content was calculated according to the following formula.

[0119] Tyrosinase activity detection: At the end of the time of action, the cells in each group were washed twice with sterile DPBS. 400 μL of 1% Triton X-100 solution was added to each well, and the cells were 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. After incubation at 37 °C for 2 h, 200 μL was pipetted into a 96-well plate, and the OD475 value was read at a wavelength of 475 nm with a microplate reader. The tyrosinase inhibition rate of each group was calculated according to the following formula.

[0120] Inhibition rate (%) = (1 - OD value of this group / OD value of UV irradiation group) × 100%.

[0121] Experimental results:

[0122] All data are expressed as mean ± standard deviation. The t-test was used for comparison between groups. P < 0.05 was indicated as *, indicating that the results had significant differences. P < 0.01 was indicated as **, indicating that the results had extremely significant differences. The results were as Figures 5 - 6 shown, where Figure 5 was the bar chart of the inhibitory effect of each group of samples on melanin content, Figure 6 was the bar chart of the inhibitory effect of each group of samples on tyrosinase activity.

[0123] As Figures 5 - 6 can be seen, ① compared with the BC group, the melanin content and tyrosinase activity in the NC group increased significantly, indicating that the UV irradiation stimulation in this experiment was effective; compared with the NC group, the melanin content and tyrosinase activity in the PC group decreased significantly, indicating that the positive control in this test was effective.

[0124] ② Compared with the NC group, in the sample A group with 3-O-ethyl ascorbic acid acetyl carnosine at a concentration of 0.02%, the sample B group with 3-O-ethyl ascorbic acid ether at a concentration of 0.01%, the sample C group with N-acetyl carnosine at a concentration of 0.01%, and the sample D group with a composition of 0.01% 3-O-ethyl ascorbic acid acetyl carnosine and 0.01% N-acetyl carnosine, based on human primary melanocytes, both the melanin content and tyrosinase activity decreased significantly. Among them, in the C group, 0.01 < p < 0.05, and both the melanin content and tyrosinase activity decreased significantly; in the A group, B group, and 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.

[0125] ③ In the sample A group with 3-O-ethyl ascorbic acid acetyl carnosine at a low concentration of 0.02%, the inhibition rate of melanin reached 21.5%, and the inhibition rate of tyrosinase activity reached 30.9%, which was significantly higher than that of the sample B group with 3-O-ethyl ascorbic acid ether (melanin inhibition rate 16.8%, tyrosinase activity inhibition rate 20.3%) and the sample C group with N-acetyl carnosine (melanin inhibition rate 8%, tyrosinase activity inhibition rate 12.4%). It was also higher than the simple mixture of 3-O-ethyl ascorbic acid ether and N-acetyl carnosine (melanin inhibition rate 19.3%, tyrosinase activity inhibition rate 27.0%). This indicates 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. While improving the stability of VC, the prepared ascorbic acid polypeptide derivatives 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.

[0126] Example 4 Photoprotective efficacy test of ascorbyl palmitate benzoyl carnosine

[0127] Oxidative stress refers to an imbalance between free radicals and the antioxidant defense system in the body. Under ultraviolet irradiation, the body will produce a large number of free radicals. Free radicals are highly reactive molecules with unpaired electrons that can initiate 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, etc. Vitamin C, as a naturally occurring antioxidant, has attracted much attention due to its strong reducing ability and extensive biological functions.

[0128] Reactive oxygen species are considered to be an important inducer of skin aging, which can lead to thinning of the skin, formation of wrinkles, etc. Ultraviolet irradiation can promote the formation of intracellular reactive oxygen species. By detecting the content of ROS in keratinocytes, the antioxidant efficacy of the sample can be reflected.

[0129] Experimental method:

[0130] (1) Cell seeding: Human primary keratinocytes were seeded into 24-well plates at a density of 5×10 4 cells / mL and incubated overnight in an incubator (37 °C, 5% CO2).

[0131] (2) Experimental grouping: The experiment was set up with a blank control group, a UV irradiation group, a positive control group, and a sample group. Each group was set with 3 replicate wells. The sample group was divided into palmitoyl ascorbate benzoyl carnosine (Group A), palmitoyl ascorbate (Group B), N-benzoyl carnosine (Group C), and a physical mixture of palmitoyl ascorbate and N-benzoyl carnosine (Group D).

[0132] Table 4: Grouping of experimental design

[0133]

[0134] (3) UV irradiation: The UV irradiation group, the positive control group, and the sample group were irradiated with UVA, and the irradiation dose was 4.8 J / cm 2 .

[0135] (4) Drug addition: After irradiation, according to the test plan in Table 4, when the cell confluence rate in the 24-well plates reached 50%, drugs were added to each group. The drug dosage per well was 1 mL, and each group had 3 replicate wells, and they were incubated in an incubator (37 °C, 5% CO2) for 24 h.

[0136] (5) Detection of ROS content: The cells in each well were washed twice with serum-free culture medium, 10 μmol / L ROS dye DCFH-DA was added, and they were incubated at 37 °C in the dark for 20 min, and then washed three times again with serum-free culture medium. Under a 20× objective lens of a fluorescence microscope, 1 field of view was randomly selected and photographed for each well, and there were 3 replicate wells in each group. After photographing, the average fluorescence intensity of ROS was calculated using Image J software, and the ROS inhibition rate was calculated according to the following formula.

[0137] ROS inhibition rate (%) = (1 - average fluorescence intensity of this group / average fluorescence intensity of the UV irradiation group) × 100%.

[0138] Experimental results:

[0139] GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis of comparisons between groups. P<0.05 was denoted as *, indicating significant differences; P<0.01 was denoted as **, indicating extremely significant differences. The results were as Figures 7 - 8 shown, among which Figure 7 was the bar graph of the inhibitory effects of each group of samples on ROS, Figure 8 and

[0140] was the fluorescence photograph of the ROS content in each group of cells. Figures 7 - 8 As

[0141] could be seen, ① compared with the BC group, the ROS content in the cells of the NC group increased significantly, indicating that the UV irradiation stimulation in this test was effective; compared with the NC group, the ROS content in the cells of the PC group decreased significantly, indicating that the positive control in this test was effective.

[0141] ② compared with the NC group, for the palmitoyl carnosine benzoyl ascorbate in the sample A group at a concentration of 10 ppm, ascorbyl palmitate in the B group at a concentration of 5 ppm, N-benzoyl carnosine in the C group at a concentration of 5 ppm, and the composition of 5 ppm ascorbyl palmitate and 5 ppm N-benzoyl carnosine in the D group, based on human primary keratinocytes, the ROS content decreased significantly. It indicated that the four groups of samples could significantly scavenge a large amount of reactive oxygen species in skin cells after ultraviolet irradiation and achieve the effect of anti-photoaging through antioxidation.

[0142] ③ At a low concentration of 10 ppm, the inhibitory rate of palmitoyl carnosine benzoyl ascorbate in the sample A group on ROS was as high as 61.5%, comparable to that of the positive control 10 mg / mL VC ethyl ether in this test (ROS inhibitory rate 60.9%), higher than that of ascorbyl palmitate in the sample B group at 5 ppm (ROS inhibitory rate 54.2%) and N-benzoyl carnosine in the sample C group at 5 ppm (ROS inhibitory rate 53.0%), and also higher than that of the simple mixture of ascorbyl palmitate and N-benzoyl carnosine at the same concentration (ROS inhibitory rate 58.6%). It showed that coupling VC palmitate with N-benzoyl carnosine could produce a powerful antioxidant effect and had a synergistic effect in inhibiting ROS generated by UV irradiation, thus achieving the effects of anti-photoaging and anti-aging, which were superior to the two raw material compounds and the physical mixture of the two raw material compounds. Therefore, the palmitoyl carnosine benzoyl ascorbate prepared in the present invention had the dual effects of antioxidation of N-benzoyl carnosine and antioxidation and whitening of VC, with low cytotoxicity, low effective concentration, and significant effect, showing broad application prospects in skin care products.

[0143] Example 5 Test on the anti-glycation effect of glucosyl ascorbate palmitoyl carnosine

[0144] Glycation refers to the non-enzymatic glycation reaction of proteins, that is, under non-enzymatic conditions, the free amino groups of macromolecules such as proteins, amino acids, lipids, or nucleic acids react with the carbonyl groups of reducing sugars through a series of reactions such as condensation, rearrangement, cleavage, and oxidative modification, ultimately forming advanced glycation end products (AGEs). AGEs are closely related to skin aging. Excessive AGEs can undergo glycation cross-linking reactions with skin elastic fibers and collagen, and AGEs are brown, ultimately causing the skin to show yellowing and reduced elasticity. Carboxymethyllysine (CML) is a structural form of AGEs. The lower the CML content, the more significant the anti-glycation effect of the sample.

[0145] Experimental method:

[0146] Inoculate human dermal fibroblasts into a 12-well plate (5×10 4 cells / well), and incubate in an incubator at 37 °C and 5% CO2 for 24 h. Remove the culture medium and gently rinse the cells with D-Hanks once or twice. Replace the blank control group with fresh culture medium, the sample group with fresh culture medium containing the modeling agent (MGO) and the sample, the model control group with fresh culture medium containing the modeling agent (MGO), and the positive control group with fresh culture medium containing the modeling agent (MGO) and aminoguanidine, and continue to incubate in the incubator for 48 h. The experimental design grouping is shown in Table 5 below:

[0147] Table 5: Experimental design grouping

[0148]

[0149] After the incubation, discard the liquid and perform washing - fixation - permeabilization - antibody incubation. Take pictures under a fluorescence microscope and use Image J software to quantitatively analyze the immunofluorescence results. Calculate the glycation inhibition rate according to the following formula.

[0150] Glycation inhibition rate (%) = (1 - average fluorescence intensity of this group / average fluorescence intensity of the model control group) × 100%.

[0151] Experimental results:

[0152] Use GraphPad Prism to plot the graph, and the results are expressed as Mean ± SD. The t-test statistical analysis is used for comparison between groups. P < 0.05 is indicated as *, indicating a significant difference; P < 0.01 is indicated as **, indicating a highly significant difference. The detection results of the anti-glycation effects of each group of samples are as Figure 9 shown.

[0153] From Figure 9It can be seen that, compared with the BC group, the average fluorescence intensity of CML in the NC group cells increased significantly, indicating that the MGO model established in this study was effective; compared with the NC group, the average fluorescence intensity of CML in the PC group cells decreased significantly, indicating that the positive control in this test was effective.

[0154] ② Compared with the NC group, for the glucosyl ascorbyl palmitoyl carnosine in the sample A group at a concentration of 0.02%, ascorbyl glucoside in the B group at a concentration of 0.01%, N-palmitoyl carnosine in the C group at a concentration of 0.01%, and the mixture of 0.01% ascorbyl glucoside and 0.01% N-palmitoyl carnosine in the D group, based on human dermal fibroblasts, the average fluorescence intensity of CML all decreased significantly. Among them, for the B group, 0.01 < p < 0.05, and the average fluorescence intensity of CML decreased significantly; for the A group, C group and D group, p < 0.01, and the average fluorescence intensity of CML decreased extremely significantly, indicating that the sample groups had obvious effects on inhibiting advanced glycation end products.

[0155] ③ At a low concentration of 0.02%, the glucosyl ascorbyl palmitoyl carnosine in the sample A group had a glycosylation inhibition rate of 43.9%, which was higher than that of 0.01% ascorbyl glucoside in the sample B group (glycosylation inhibition rate of 14.2%) and 0.01% N-palmitoyl carnosine in the sample C group (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 indicates 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 actions of the two raw material compounds and the physical mixture of the two raw material compounds.

[0156] Therefore, the glucosyl ascorbyl N-palmitoyl carnosine prepared by the present invention has the dual effects of N-palmitoyl carnosine's anti-glycation, anti-oxidation and VC's anti-oxidation and skin whitening, can effectively delay skin aging, help whiten the skin, and improve the problem of uneven skin tone.

[0157] Example 6 Stability experiment of ascorbic acid polypeptide derivatives

[0158] Experimental method:

[0159] 3 - O - ethyl ascorbyl acetyl carnosine (Formula 1), 3 - O - acetyl ascorbyl acetyl carnosine (Formula 7), ascorbyl glucoside acetyl carnosine (Formula 13), ascorbyl palmitate acetyl carnosine (Formula 16), and the prototype L - ascorbic acid were respectively prepared into aqueous solutions with a concentration of 50 mg / L, placed in 50 mL glass bottles, and stored 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, 400 μL of the solution was taken from each bottle and poured into a quartz cuvette, and its absorbance was measured using an ultraviolet - visible spectrophotometer. Each group was measured in parallel three times. The stability was evaluated by measuring the decrease in absorbance at the maximum absorption wavelength of each group of samples. The stability calculation formula is as follows:

[0160] Stability (%) = Absorbance on day n / Absorbance on day 0 × 100%.

[0161] Experimental results:

[0162] GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The stability of each group of samples under the condition of 43 °C in the dark is as Figure 10 shown.

[0163] From Figure 10 it can be seen that: ① Only about 20% of the prototype ascorbic acid remained after 1 day, and only 8% remained 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 relatively active in chemical reactions and is prone to electron transfer or oxidation. And it contains more free - OH in its molecule, with high activity, and is prone to react with other substances. Especially under aerobic conditions, these hydroxyl groups are easily oxidized. The stability of ascorbic acid polypeptide derivatives within 10 days has been improved compared with the prototype ascorbic acid. It is speculated that this is because ascorbic acid polypeptide derivatives reduce the activity of the enol - type hydroxyl groups in the ascorbic acid molecule by changing their chemical structure, thereby reducing the possibility of being oxidized by light, heat, and air.

[0164] ② Among the four derivatives, the stability of 3 - O - ethyl ascorbyl acetyl carnosine is the best, with more than 90% integrity remaining after 10 days. The stability of 3 - O - acetyl ascorbyl acetyl carnosine is the second (88% remaining after 10 days), and that of ascorbyl palmitate acetyl carnosine is the third (74% remaining after 10 days). The stability of ascorbyl glucoside acetyl carnosine is lower than that of the other three derivatives, probably because it has more free hydroxyl groups and the sugar ring is more easily degraded. However, its remaining amount after 10 days (28%) is still higher than that of the prototype VC (8%), indicating that the conjugate of VC derivative and carnosine derivative improves the stability of VC, greatly expanding the application scope of ascorbic acid polypeptide derivatives in the fields of cosmetic formulations and pharmaceutical preparations.

[0165] Example 7 Comparison of the Whitening Effects of Ascorbic Acid Polypeptide Derivatives

[0166] Experimental Purpose:

[0167] The 3-O-ethylascorbic acid acetyl carnosine (Formula 1), 3-O-ethylascorbic acid palmitoyl carnosine (Formula 3), and 3-O-ethylascorbic acid benzoyl carnosine (Formula 5) provided by the present invention were compared with the 3-O-ethylascorbic acid carnosine described in Chinese Patent CN 115260170 B in terms of whitening effect on a melanocyte model (at the same concentration), so as to compare the differences in the whitening effect of the combination of different carnosine derivatives and ascorbic acid ethyl ether.

[0168] Synthesis of 3-O-ethylascorbic acid carnosine:

[0169] Compared with Chinese Patent CN 115260170 B, the present invention improved the synthesis process of 3-O-ethylascorbic acid carnosine, and the specific steps are as follows:

[0170] 。

[0171] Step 1: Synthesis of N-Boc-carnosine

[0172] 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 it in a 30°C water bath, slowly dropwise add 11 g of di-tert-butyl dicarbonate, and after the addition, react for 5 h. Cool the reaction solution to 0°C, maintain this temperature and dropwise add 2 mol / L hydrochloric acid to adjust the system pH = 2, and extract with ethyl acetate (20 mL × 4). 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 white solid appears, add a large amount of petroleum ether and stir. After filtration, N-Boc-carnosine is obtained.

[0173] Step 2: Activation of N-Boc-carnosine

[0174] Dissolve 3.26 g of N-Boc-carnosine (10 mmol) obtained in Step 1 in 100 mL of dichloromethane, add 2.06 g of DCC (N,N'-dicyclohexylcarbodiimide, 10 mmol) and 1.22 g of DMAP (4-dimethylaminopyridine, 10 mmol), and stir at room temperature for 10 h. After the reaction is completed, filter to remove the by-product DCU (N,N'-dicyclohexylurea) to obtain a filtrate.

[0175] Step 3: Synthesis of 3-O-ethyl ascorbic acid-N-Boc-carnosine

[0176] 2.04 g of 3-O-ethyl ascorbic acid ether (Formula 22, 10 mmol) and 2.02 g of the acid-binding agent triethylamine (20 mmol) were added to the filtrate of Step 2, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, it was concentrated under reduced pressure using a rotary evaporator and then dried in a vacuum drying oven. The solid substance was recrystallized from 20 mL of isopropanol at 50 °C, filtered, and the solid remaining on the filter paper was taken to obtain 3-O-ethyl ascorbic acid-N-Boc-carnosine.

[0177] Step 4: Synthesis of 3-O-ethyl ascorbic acid carnosine

[0178] A mixed solution of 18 mL of trifluoroacetic acid and 20 mL of dichloromethane was added to all the products obtained in Step 3, and the mixture was stirred at room temperature for 1 h to remove the N-Boc protecting group. The reaction was stopped, the solvent was removed by concentration using a rotary evaporator, and the remaining product was dissolved in 10 mL of ethyl acetate. Then, it was washed by stirring with 20 mL of 5 wt% Na2CO3 solution until the pH reached 8.5; then, it was allowed to stand for liquid separation, the organic phase was sucked out with a pipette and concentrated using a rotary evaporator to remove ethyl acetate, and then freeze-dried to obtain white powder 3-O-ethyl ascorbic acid carnosine (Formula 27).

[0179] The structure of 3-O-ethyl ascorbic acid carnosine prepared according to the above method of the present invention is shown in Formula (27), and its 1H NMR structure identification is as follows:

[0180] 1H NMR of formula (27): 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); Total yield 24.5%.

[0181] 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 of 4.9% recorded in paragraph 154 of its specification).

[0182] In vitro whitening efficacy test:

[0183] (1) Cell seeding: Human primary melanocytes were seeded into a 24 - well plate at a density of 5×10 4 cells / mL and incubated overnight in an incubator (37°C, 5% CO2).

[0184] (2) Experimental grouping: The experiment was set up with a blank control group, a UV irradiation group, a positive control group, and a sample group. Each group was set with 3 replicate wells.

[0185] Table 6: Grouping of experimental design

[0186]

[0187] (3) UV irradiation: The UV irradiation group, the positive control group, and the sample group were irradiated with UVA, and the irradiation dose was 4.8 J / cm 2 .

[0188] (4)Drug addition: According to the test plan in Table 6, when the cell seeding rate in the 24-well plate reaches 50%, group-administer the drug. The drug dosage per well is 1 mL, and 3 replicates are set for each group. Incubate in an incubator (37 °C, 5% CO2) for 24 h.

[0189] (5)The detection methods for melanin content and tyrosinase activity are the same as those in Example 3.

[0190] Experimental results:

[0191] All data are expressed as mean ± standard deviation. The t-test is used for comparison between groups. P < 0.05 is denoted as *, indicating that the results have significant differences. P < 0.01 is denoted as **, indicating that the results have extremely significant differences. The results are as Figures 11 - 12 shown, where Figure 11 is the bar graph of the inhibitory effect of each group of samples on melanin content, Figure 12 and

[0192] is the bar graph of the inhibitory effect of each group of samples on tyrosinase activity. Figures 11 - 12 As can be seen from

[0193] Example 8 Anti-aging human efficacy of the essence containing ascorbic acid polypeptide derivatives

[0194] (1)Preparation of anti-wrinkle essence: 4% 1,3-butanediol, 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 the base essence. The products synthesized in Example 1, 3-O-ethylascorbic acid acetyl carnosine (Formula 1, 10 ppm), 3-O-ethylascorbic acid palmitoyl carnosine (Formula 3, 10 ppm), 3-O-ethylascorbic acid benzoyl carnosine (Formula 5, 10 ppm) were added to the base essence as an anti-aging composition, as Group A of the samples. 3-O-ethylascorbic acid 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 Group B of the samples.

[0195] (2)Selection of volunteers: The selection of subjects follows the medical and ethical standards of human testing. All tests on subjects must be voluntary by the subjects themselves, and an informed consent form must be signed before the test. 60 healthy female subjects, aged 18 - 40 years old, were recruited and randomly divided into 2 groups, with 30 people in each group.

[0196] (3)Usage method of experimental samples: After cleansing their faces in the morning and evening every day, the two groups of subjects used the essence of Group A and Group B for skin care respectively. Each time, 0.2 g of the essence was used and gently massaged until absorbed. Other anti-wrinkle skin care products were discontinued during the experiment.

[0197] (4)Before using the samples and 14 days and 28 days after using the samples, the subjects' facial images were collected using VISIA-CR. The subjects' images were collected using VC20 Plus and the cheek skin roughness SEr and skin smoothness SEsm were analyzed. Each probe of the German CK instrument was used: Corneometer CM 825 was used to measure the moisture content of the cheek stratum corneum, Glossymeter CL200 was used to measure the skin gloss, and Colorimeter CL400 was used to measure the skin color. Using SPSS analysis software, the measured values at different time points were compared with the baseline values before using the samples, and the Shapiro-Wilk Test was used to conduct a significance test on the normal distribution of the data improvement values.

[0198] Before the test, the subjects were asked to wash their faces with the same cleansing product, and were stabilized in a stable room at a constant temperature and humidity (temperature: 21 °C ± 1 °C, humidity: 50% ± 10%) for 20 min to keep the skin state stable, and then the above detections were carried out.

[0199] (5)The results are shown in the following table:

[0200] The change rate after using the product = (data after use - data before use) / data before use × 100%.

[0201] Table 7: Test Results of Each Skin Parameter before and after Using the Anti-Wrinkle Serum

[0202]

[0203] According to the data in Table 7, after 14 days and 28 days of using the serum containing 3-O-ethylascorbic acid acetyl carnosine, 3-O-ethylascorbic acid palmitoyl carnosine, and 3-O-ethylascorbic acid benzoyl carnosine, the water content of the skin stratum corneum, skin gloss, and elasticity of the subjects were significantly improved; while the average volume and area ratio of cheek wrinkles decreased significantly, and skin flushing was also significantly improved. Moreover, the improvement of the parameters of each skin index of the subjects using Sample A was better than that of Sample B disclosed in Patent CN 115260170 B. The above results indicate that the serum containing 3-O-ethylascorbic acid acetyl carnosine, 3-O-ethylascorbic acid palmitoyl carnosine, and 3-O-ethylascorbic acid benzoyl carnosine provided by the present invention has the effects of moisturizing and locking water, reducing skin wrinkles, improving skin gloss, enhancing skin elasticity, and repairing skin flushing, making the skin look more energetic and younger in appearance, and having significant anti-aging and repair effects.

[0204] 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 equivalent technologies, the present invention is also intended to include these changes and modifications.

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 formulas (1), (3), (5), (14), and (18); Wherein, formula (1) is the product of coupling 3-O-ethyl ascorbic acid ether with N-acetylcarnosine; Formula (3) is the product of coupling 3-O-ethyl ascorbic acid ether with N-palmitoyl carnosine; Formula (5) is the product of coupling 3-O-ethyl ascorbic acid ether with N-benzoylcarnosine; Formula (14) is the product of the coupling of ascorbyl glucoside 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; ； Raw material B is an ascorbic acid derivative, as shown in any one of the structures of formula (22), (24) and (25); wherein formula (22) is 3-O-ethyl 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; 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 any one of formulas (1), (3), (5), (14), and (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.

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

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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