WO2022228475A1 - 抗坏血酸多肽衍生物及其制备方法和应用 - Google Patents

抗坏血酸多肽衍生物及其制备方法和应用 Download PDF

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WO2022228475A1
WO2022228475A1 PCT/CN2022/089635 CN2022089635W WO2022228475A1 WO 2022228475 A1 WO2022228475 A1 WO 2022228475A1 CN 2022089635 W CN2022089635 W CN 2022089635W WO 2022228475 A1 WO2022228475 A1 WO 2022228475A1
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ascorbic acid
acid polypeptide
polypeptide derivative
group
carnosine
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PCT/CN2022/089635
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English (en)
French (fr)
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李钧翔
徐�明
陆益
康思宁
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禾美生物科技(浙江)有限公司
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Priority to US18/557,911 priority Critical patent/US20240261204A1/en
Publication of WO2022228475A1 publication Critical patent/WO2022228475A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/676—Ascorbic acid, i.e. vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present disclosure relates to the technical field of cosmetic raw material development, in particular to an ascorbic acid polypeptide derivative and a preparation method and application thereof.
  • UVA is known as the invisible killer of skin aging. Its damage to the skin is very subtle and has no obvious signs, but the damage is irreversible. UVA can not only cause melanosis and make the skin “dark”, but also induce the production of ROS, causing the loss of collagen, making the skin gradually age, wrinkles, sagging and dark dullness. Therefore, simultaneously resisting melanin deposition and aging at the level of dermal cells is an efficient way of anti-aging.
  • L-Ascorbic acid is a natural water-soluble antioxidant for whitening, also known as vitamin C, which is widely used as an anti-aging ingredient in cosmetics.
  • vitamin C also known as vitamin C
  • it is sensitive to light and oxygen, and its unstable structure limits its use. Application in the cosmetic industry.
  • One of the purposes of the present disclosure is to provide an ascorbic acid polypeptide derivative with a new structure, which combines the excellent effects of VC whitening and carnosine anti-aging, and solves the stability problem of the traditional VC structure.
  • the second purpose of the present disclosure is to provide a preparation method of ascorbic acid polypeptide derivatives.
  • the third purpose of the present disclosure is to provide an application of an ascorbic acid polypeptide derivative in the preparation of cosmetics.
  • the fourth object of the present disclosure is to provide a cosmetic comprising an ascorbic acid polypeptide derivative as an active ingredient.
  • the present disclosure provides an ascorbic acid polypeptide derivative, a compound represented by the following general formula (1) or a salt thereof:
  • X 1 and X 2 are independently a C1-C6 alkyl group, a C1-C6 alkoxy group or a halogen group;
  • Q 1 and Q 2 are independently C1-C6 alkyl chains
  • a, b, and c are independently an integer selected from 1-10;
  • e is 0 and d is an integer from 1 to 10, or, e is 1 and d is 0.
  • the ascorbic acid polypeptide derivative is a compound represented by the following general formula (3):
  • the ascorbic acid polypeptide derivative is a compound represented by the following general formula (4):
  • the ascorbic acid polypeptide derivative is a compound represented by the following general formula (6):
  • the present disclosure provides a preparation method of the above-mentioned ascorbic acid polypeptide derivative, comprising:
  • the carboxyl group is activated on the raw material A with the amino protective group, and then reacted with the raw material B, and the ascorbic acid polypeptide derivative is obtained after deprotecting the protective group;
  • the raw material A is a compound represented by the following general formula (7):
  • a and b are independently selected from integers from 1 to 10;
  • the raw material B is a compound represented by the following general formula (8):
  • k is selected from an integer from 1 to 10;
  • Q is an alkyl chain of C1 ⁇ C6
  • Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, or a halogen group.
  • the raw material A is carnosine with an amino protecting group; the raw material B is 3-O-ethyl ascorbic acid;
  • the preparation method comprises the following steps:
  • the raw material A with the amino protecting group is first reacted with dicyclohexylcarbodiimide and N-hydroxysuccinimide in an organic solvent, after the reaction is completed, the by-products are removed, and then the raw material B is added to react with an acid binding agent, and then the reaction is removed. protective group to obtain the ascorbic acid polypeptide derivative.
  • the present disclosure provides an application of the above-mentioned ascorbic acid polypeptide derivative in the preparation of cosmetics.
  • the present disclosure provides a cosmetic comprising the above-mentioned ascorbic acid polypeptide derivative as an active ingredient; the effective concentration of the ascorbic acid polypeptide derivative is 10-50 ppm.
  • the present disclosure couples the ascorbic acid derivative structure and the polypeptide structure, and designs and synthesizes a new ascorbic acid polypeptide derivative by molecular modification and modification, and the chemical structure is clear and definite.
  • the new structure of ascorbic acid polypeptide derivative has stable structure and high biological safety, which solves the problem of unstable structure of traditional VC.
  • the related ascorbic acid derivatives obtained in the present disclosure also have anti-glycation, anti-blue light, anti-autophagy and other effects, and can be used on the skin of various parts (hands, face, limbs, scalp, etc.).
  • Fig. 1 is the synthetic route diagram of the 3-O-ethyl ascorbic acid-carnosine of embodiment 1;
  • Fig. 2 is the HPLC spectrogram of 3-O-ethylascorbic acid-carnosine synthesized in Example 1;
  • Fig. 3 is the MS spectrum of 3-O-ethylascorbic acid-carnosine synthesized in Example 1;
  • Fig. 4 is the hydrogen nuclear magnetic resonance spectrum of 3-O-ethyl ascorbic acid-carnosine synthesized in Example 1;
  • Fig. 5 is the H, H-COSY spectrum of 3-O-ethyl ascorbic acid-carnosine synthesized in Example 1;
  • Figure 6 is a graph showing the results of the SIRC corneal cell stimulation test in Test Example 1;
  • Fig. 7 is Test Example 1 corrosive chicken embryo chorioallantoic membrane test result diagram
  • Figure 8 is a graph of the results of the tyrosinase enzyme activity test of Test Example 2;
  • Fig. 9 is Test Example 2 melanin test result diagram, wherein (a) is the test result under different raw materials, and (b) is the test result under different concentrations;
  • FIG. 10 is a graph showing the results of the test of the energy metabolism ability of keratinocytes in Test Example 2;
  • Figure 11 is a graph showing the results of an anti-aging repair test of stratum corneum cells under oxidative stress in Test Example 2;
  • Figure 12 is a graph showing the results of the test for preventing UVA-induced damage on immortalized keratinocytes (HaCaT) in Test Example 2, wherein (a) is the sample cytotoxicity detection graph; (b) is the cell viability (CSR) after preventing UVA damage; ( c) is the expression of intracellular reactive oxygen species (ROS) after preventing UVA damage; (d) is the expression of intracellular hydroxyproline (HYP) after preventing UVA damage;
  • Figure 13 is a graph showing the results of a synthesis test of type I collagen in fibroblasts of Test Example 2;
  • Figure 14 is a graph showing the results of the test for preventing UVA-induced damage by human foreskin fibroblasts (HFF) in Test Example 2, wherein (a) is the cell viability (CSR) after preventing UVA damage; (b) is the intracellular level after preventing UVA damage. Reactive oxygen species (ROS) expression; (c) Intracellular hydroxyproline (HYP) expression after UVA injury prevention;
  • CSR cell viability
  • ROS Reactive oxygen species
  • HFF Intracellular hydroxyproline
  • Figure 15 is a graph showing the results of collagen I content in HFF cells under UVA-induced injury
  • Fig. 16 is the result graph of the skin elasticity value of the test group of Test Example 3.
  • FIG. 17 is a graph showing the results of ITA skin color and lustre of the test group in Test Example 3.
  • FIG. 17 is a graph showing the results of ITA skin color and lustre of the test group in Test Example 3.
  • This new ascorbic acid peptide not only exhibits stable properties in extracellular matrix enhancement, antioxidant and melanin inhibition, but also has significantly improved anti-aging properties compared to ascorbic acid and peptides.
  • an ascorbic acid polypeptide derivative a compound represented by the following general formula (1) or a salt thereof:
  • Ascorbic acid polypeptide derivative is a stable derivative formed by organic (chemical bond) combination of ascorbic acid and polypeptide.
  • Compound salts generally refer to metal salts of compounds, such as sodium salts, potassium salts, and the like.
  • a is selected from an integer of 1-10, and a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • b is selected from the integer of 1-10, b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • e is 0 and d is an integer from 1 to 10 (d is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), or, e is 1 and d is 0;
  • Q 2 is a C1-C6 alkyl chain, which can be a straight chain or a branched chain;
  • X 2 is a C1-C6 alkyl group, a C1-C6 alkoxy group or a halogen group;
  • Alkoxy groups include, but are not limited to, methoxy, ethoxy, acetoxy, isopropoxy, isopropenyloxy, tert-butoxy, tert-butylperoxy, or butanone oxime;
  • Alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, or butyl (n-butyl, isobutyl, sec-butyl, tert-butyl).
  • Halogen groups include, but are not limited to, chloro, bromo, iodo.
  • an alkyl group includes a linear or branched alkyl group.
  • X 2 is ethoxy
  • the ascorbic acid polypeptide derivative of (I) is a compound represented by the following general formula (2) or a salt thereof:
  • R 1 is a C1-C6 alkyl group, including but not limited to methyl, ethyl, propyl, isopropyl or butyl (n-butyl, isobutyl, sec-butyl, tert-butyl); Radical includes straight or branched chain alkyl.
  • a, b are independently an integer of 1-5 (a, b are independently 1, 2, 3, 4 or 5);
  • e is 0 and d is an integer from 1 to 5 (d is 1, 2, 3, 4, or 5), or, e is 1 and d is 0.
  • R 1 is an ethyl group
  • Q 2 is a C2 alkyl chain
  • a is 2
  • b is 1
  • e is 1
  • d is 0, and the ascorbic acid polypeptide derivative is represented by the following general formula (3) Representative compounds:
  • R 1 is an ethyl group
  • Q 2 is a C1 alkyl chain
  • a is 2
  • b is 1
  • e is 0
  • d is 1
  • the ascorbic acid polypeptide derivative is composed of the following general formula (4 ) represents the compound:
  • a is selected from an integer of 1-10, and a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • b is selected from the integer of 1-10, b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • c is selected from an integer of 1-10, c is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • Q 1 is a C1-C6 alkyl chain, which can be a straight chain or a branched chain;
  • X 1 is a C1-C6 alkyl group, a C1-C6 alkoxy group or a halogen group;
  • Alkoxy groups include, but are not limited to, methoxy, ethoxy, acetoxy, isopropoxy, isopropenyloxy, tert-butoxy, tert-butylperoxy, or butanone oxime;
  • Alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl or butyl (n-butyl, isobutyl, sec-butyl, tert-butyl), including straight or branched chain alkyl groups .
  • Halogen groups include, but are not limited to, chloro, bromo, iodo.
  • X 1 is ethoxy
  • the ascorbic acid polypeptide derivative of (II) is a compound represented by the following general formula (5) or a salt thereof:
  • R 2 is a C1-C6 alkyl group, including but not limited to methyl, ethyl, methylethyl, propyl, isopropyl or butyl (n-butyl, isobutyl, sec-butyl, tertiary Butyl);
  • Q 1 is a C1-C6 alkyl chain
  • a, b, c are independently an integer of 1-5 (a, b, d are independently 1, 2, 3, 4 or 5).
  • Q 1 is a C1 alkyl chain
  • R 2 is an ethyl group
  • a is 2
  • b and c are independently 1
  • the ascorbic acid polypeptide derivative is a compound represented by the following general formula (6):
  • the present disclosure also provides a preparation method of the above-mentioned ascorbic acid polypeptide derivative, comprising:
  • the carboxyl group is activated on the raw material A with the amino protective group, and then reacted with the raw material B, and the ascorbic acid polypeptide derivative is obtained after deprotecting the protective group;
  • the raw material A is a compound represented by the following general formula (7):
  • p is selected from the integer of 1-10, p is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • q is selected from the integer of 1-10, q is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • Raw material B is a compound represented by the following general formula (8):
  • k is selected from an integer of 1-10, and k is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • Q is an alkyl chain of C1 ⁇ C6
  • Y is a C1-C6 alkyl group, a C1-C6 alkoxy group, or a halogen group.
  • Alkoxy groups include, but are not limited to, methoxy, ethoxy, acetoxy, isopropoxy, isopropenyloxy, tert-butoxy, tert-butylperoxy, or butanone oxime;
  • Alkyl groups include, but are not limited to, methyl, ethyl, methylethyl, propyl, isopropyl, or butyl (n-butyl, isobutyl, sec-butyl, tert-butyl), including straight chain or branched chain alkyl.
  • Halogen groups include, but are not limited to, chloro, bromo, iodo.
  • starting material A is carnosine with amino protecting group (L-Carnosine):
  • Raw material B is 3-O-ethyl ascorbic acid (EAC):
  • the compound represented by general formula (3), (4), (6) or its salt can be prepared according to the following reaction:
  • the preparation method comprises the following steps:
  • Carnosine with an amino protecting group is first reacted with dicyclohexylcarbodiimide and N-hydroxysuccinimide in an organic solvent. After the reaction, by-products are removed, and then 3-O-ethyl ascorbic acid and acid binding agent are added. After the reaction, the protective group is deprotected to obtain ascorbic acid polypeptide derivatives.
  • Carnosine carboxyl groups were activated with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS).
  • DCC dicyclohexylcarbodiimide
  • NHS N-hydroxysuccinimide
  • Organic solvents include, but are not limited to, DMF, dioxane (1,4-dioxane), acetone.
  • Acid binding agents include, but are not limited to, N,N-diisopropylethylamine, diisopropylethylamine (EDIPA), triethylamine.
  • the reaction temperature and reaction time can be generally 1-30h, optionally 12-18h, and the reaction temperature can generally be 5-50°C, optionally 15-35°C.
  • the preparation method is as follows:
  • the present disclosure also provides an application of the above-mentioned ascorbic acid polypeptide derivative in the preparation of cosmetics.
  • the present disclosure also provides a cosmetic comprising the above-mentioned ascorbic acid polypeptide derivative as an active ingredient; the effective concentration of the ascorbic acid polypeptide derivative is 10-50 ppm.
  • the compounds of the present disclosure effectively combine the ascorbic acid derivative structure and the polypeptide structure (typically the functional components vitamin VC (L-ascorbic acid) and carnosine ( ⁇ -alanyl-L-histidine), and rationalize the molecules
  • the modified modified design was finally developed.
  • the ascorbic acid polypeptide derivative with a new structure has both the excellent effects of VC whitening and carnosine anti-aging, and solves the stability problem of the traditional VC structure.
  • the safety and efficacy performance of the innovative polypeptide derivatives were evaluated through various cell, semi-in vivo models and population efficacy evaluations, which proved that good whitening and anti-aging synergy can be obtained at a lower effective concentration (24.25 ⁇ M/10ppm).
  • Efficacy including stimulation of collagen synthesis by carnosine and L-ascorbic acid, inhibition of ROS and melanin production, etc.
  • Cosmetic samples were prepared through scientific formulations, and reasonable volunteer efficacy tests were carried out to verify the anti-aging activity of the compound, which proved that (10-50 ppm) ascorbic acid polypeptide derivatives with new structures can be used in cosmetics under the condition of economical conditions. usage scenarios.
  • Reagents used DCC, C 13 H 22 N 2 , dicyclohexylcarbodiimide, dehydrating agent; NHS, N-Hydroxysuccinimide, also known as HOSu, C 4 H 5 NO 3 , N-hydroxysuccinimide; DMF , dimethylformamide, water absorbent, miscible with most organic solvents; DIPEA, N,N-diisopropylethylamine, basic organic solvent; 1,4-dioxane, 1,4 dioxane, Solvent, stabilizer.
  • DCC C 13 H 22 N 2 , dicyclohexylcarbodiimide, dehydrating agent
  • NHS N-Hydroxysuccinimide, also known as HOSu, C 4 H 5 NO 3 , N-hydroxysuccinimide
  • DMF dimethylformamide, water absorbent, miscible with most organic solvents
  • DIPEA N,N-diisopropylethylamine, basic
  • Boc-L-Carnosine 13g, 39.9mmoL was dissolved in DMF (400mL);
  • Example 1 The substance synthesized in Example 1 was used as a cosmetic raw material, and various cell models were used to evaluate its safety and efficacy, including the biosafety evaluation of in vitro corneal cells (SIRC), and the evaluation of antioxidant activity of human keratinocytes (HaCaT) ROS Content reduction test, assessment of collagen synthesis in human skin fibroblasts (FB) and assessment of tyrosinase activity and melanin regulation of melanoma cell (B16) model. In vivo studies for validation of anti-aging effects were also performed using the VISIA CR and CK instruments.
  • SIRC in vitro corneal cells
  • HaCaT human keratinocytes
  • FB human skin fibroblasts
  • B16 melanin regulation of melanoma cell
  • Example 1 The substances synthesized in Example 1 have completed the following safety animal substitution tests under the company's own cell model platform, as shown in the following table:
  • Detection method Rabbit corneal cell SIRC cell viability test, wash the cells to be tested with PBS for 1 to 2 times, add 100 ⁇ L of MEM complete medium and 10 ⁇ L of CCK-8 solution to each well, and place them in a carbon dioxide incubator for 2 to 4 hours. . At a wavelength of 450 nm, the absorbance (OD) value was measured. The CCK-8 reagent can be replaced with MTT.
  • the cell samples treated with the inducer ( ⁇ -MSH) and the test substance were placed in a 96-well cell culture plate for repeated freeze-thaw lysis, and 100 ⁇ L of 0.1-0.5% levodopa was added to each well, and the cell incubator was 37°C. Incubate for about 2h, and measure the absorbance (OD) value at a wavelength of 450nm. The results are shown in Figure 8.
  • the cell samples treated with the inducer ( ⁇ -MSH) and the analyte were placed in a 6-well cell culture plate, 0.5-2 mL of cell lysate was added, and placed in a constant temperature oven at 65 °C for 2 h. Then, after mixing the lysate, 150 ⁇ L/well was added to a 96-well assay plate, and the absorbance (OD) value was measured at a wavelength of 450 nm. The results are shown in Figure 9.
  • EAC-carnosine has a significant whitening effect, and in some indicators is better than the gold standard ingredients such as 3-O-Ethyl Ascorbic Acid (3-O-Ethyl Ascorbic Acid) and 4-methoxysalicylic acid potassium (4- MSK), etc.
  • EAC-L-Carnosine at 24.25 ⁇ M can significantly inhibit the secretion of melanin. At the concentration of 24.25 ⁇ M, the effect of EAC-L-Carnosine was the best. It is speculated that it may be related to the reduction, carnosine structure and cell proliferation of EAC-L-Carnosine.
  • Human immortalized keratinocyte HaCaT after plating, when the cell confluence is about 65%, remove the original medium, use medium containing a certain concentration of H 2 O 2 , and place it in a carbon dioxide incubator for 2h. After induction, the original medium was removed, replaced with a medium containing the sample to be tested (or positive sample), and placed in a carbon dioxide incubator.
  • ROS detection After 24 hours, replace with a certain amount of DCFH-DA working solution, incubate in the dark for 15-30 minutes, wash with serum-free medium for 3-5 times, add 100 ⁇ L of serum-free medium, excitation wavelength at 488nm, emission wavelength at 525 , to detect the fluorescence intensity after stimulation.
  • Detection of cell viability After 24h, replace with serum-free medium containing 10% CCK-8, and after 2-4h incubation in a carbon dioxide incubator, detect the OD value at a wavelength of 450nm.
  • FIG. 11 shows the expression of ROS in immortalized keratinocytes (HaCaT) after the samples were repaired for 24 hours after hydrogen peroxide (H 2 O 2 ) injury.
  • Human immortalized keratinocyte HaCaT after plating, when the cell confluence is about 65%, remove the original medium, replace the original medium with PBS containing the sample to be tested, and place it under a specific ultraviolet device, UVA irradiation dose is 15J/cm 2 . After induction, the drug-containing PBS was removed, replaced with a medium containing the sample to be tested, and placed in a carbon dioxide incubator.
  • ROS detection After 24 hours, replace with a certain amount of DCFH-DA working solution, incubate in the dark for 15-30 minutes, wash with serum-free medium for 3-5 times, add 100 ⁇ L of serum-free medium, excitation wavelength at 488nm, emission wavelength at 525 , to detect the fluorescence intensity after stimulation.
  • Detection of cell viability After 24h, replace with serum-free medium containing 10% CCK-8, and after 2-4h incubation in a carbon dioxide incubator, detect the OD value at a wavelength of 450nm.
  • a certain dose of UVA-induced damage to HaCaT was selected to construct a UVA-damaged cell model of keratinocytes.
  • Figure 12 is a graph of the experimental data of UVA damage in HaCaT cells, and the conclusions are as follows:
  • EAC-carnosine (about 24 ⁇ M/10 ppm) of the present disclosure up-regulates the activity of keratinocytes after UVA irradiation, significantly inhibits the generation of free radicals ROS, and inhibits collagen precursor hydroxyproline without cytotoxicity Acid HYP has a significant activating effect.
  • the above index levels were better than the effect of VC monomer (24 ⁇ M), and were close to the positive control TGF- ⁇ 1 (100ng/mL).
  • Human primary fibroblast FB after plating, the cell confluence is 45% to 65%, remove the original medium, replace the original medium with PBS containing the sample to be tested, and place it in a carbon dioxide incubator for 24h ⁇ 2h. After incubation, 200 ⁇ L of cell culture supernatant was collected from each well in a 1.5 mL sterile centrifuge tube, and placed in a -80°C ultra-low temperature freezer for cryopreservation.
  • TGF-B1 was the positive control with a concentration of 100ng/mL. It can be seen that EAC-carnosine can significantly enhance the synthesis of type I collagen in fibroblasts.
  • HFF human foreskin fibroblasts after plating, when the cell confluence is 65% to 85%, remove the original medium, replace the original medium with PBS containing the sample to be tested, place it under a specific UV device, and irradiate with UVA The dose was 5 J/cm 2 . After induction, the drug-containing PBS was removed, replaced with a medium containing the sample to be tested, and placed in a carbon dioxide incubator.
  • ROS detection After 24 hours, replace with a certain amount of DCFH-DA working solution, incubate in the dark for 15-30 minutes, wash with serum-free medium for 3-5 times, add 100 ⁇ L of serum-free medium, excitation wavelength at 488nm, emission wavelength at 525 , to detect the fluorescence intensity after stimulation.
  • Detection of cell viability After 24h, replace with serum-free medium containing 10% CCK-8, and after 2-4h incubation in a carbon dioxide incubator, detect the OD value at a wavelength of 450nm.
  • HYP (cell lysate) content detection use the hydroxyproline (HYP) content detection kit. First, use the HYP standard to detect and draw a standard curve, and then detect the content of HYP (cell supernatant) contained in the fibroblast lysate diluted in an appropriate ratio. The results are shown in Figure 14.
  • Type I collagen content detection ELISA detection, using human type I collagen (Col I) enzyme-linked immunosorbent assay kit. First, the collagen type I standard was used to detect and draw a standard curve, and then, the content of collagen contained in the fibroblast supernatant diluted in an appropriate ratio was detected.
  • EAC-carnosine (about 24 ⁇ M/10 ppm), without cytotoxicity, significantly up-regulates the activity of fibroblasts after UVA irradiation, significantly inhibits the generation of free radicals ROS, and inhibits the collagen precursor hydroxyproline HYP has a significant activation effect.
  • the above index levels are superior to the synergistic effect of the mixed addition of VC monomer/EAC monomer and carnosine, as well as the effect of the patented VC derivative AA2G, and are close to the positive control TGF- ⁇ 1.
  • EAC-carnosine has a significant anti-aging and repairing effect on multiple multi-dimensional indicators, and some indicators are better than the simple superposition of the effects of the classic physical mixing of carnosine and VC derivatives, with a synergistic anti-aging effect, achieving "1" +1>2" performance.
  • Test method 20ppm EAC-dipeptide raw material was added to the base cream, and used twice a week, and the effect was tested at 4 weeks and 8 weeks, with the base cream without EAC-dipeptide raw material as a blank placebo.
  • Test population 15 experimental group, 15 placebo group. (31-54 years old, female).
  • Test equipment CK instrument.
  • Test method Using the cutometer and Colorimeter probe of the CK equipment, under the constant temperature and humidity environment, the skin elasticity test and the skin color test were carried out on each volunteer's random cheek after 0 weeks, 4 weeks and 8 weeks of product use;
  • the results of the skin elasticity value of the experimental group are shown in Figure 16; the results show that: the skin elasticity value R2 of the experimental group has a significant increase trend on the 14th and 28th days; the increase in the placebo group is lower than that in the experimental group, which proves that Significant applied anti-aging efficacy of EAC-dipeptide.

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Abstract

本公开提供了一种抗坏血酸多肽衍生物及其制备方法和应用，涉及化妆品原料开发技术领域，抗坏血酸多肽衍生物为由通式(1)代表的化合物或其盐。本公开全新结构的抗坏血酸多肽衍生物，兼具了VC美白和肌肽抗衰的优异功效，并解决了传统VC结构的稳定性问题。细胞实验证明，化合物具有良好的生物活性，包括降低ROS，促进胶原蛋白I的产生，降低酪氨酸酶的活性和下调黑色素的分泌。在24.25μM的浓度下，EAC-L-肌肽可将黑色素的分泌下调27.85％，大于相同浓度下的EAC和肌肽的总和。在皮肤弹性，皮肤色调和减少皱纹方面具有理想的抗衰老作用，是一种有前途的抗衰老活性物质。

Description

抗坏血酸多肽衍生物及其制备方法和应用
相关申请的交叉引用
本公开要求于2021年04月30日提交中国专利局的申请号为“CN 202110488537.7”名称为“抗坏血酸多肽衍生物及其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及化妆品原料开发技术领域，尤其是涉及一种抗坏血酸多肽衍生物及其制备方法和应用。
背景技术
随着地球臭氧层不断破坏，紫外线日益强烈，人们愈发重视预防“光老化”的方法和原料。UVA被称为肌肤老化的隐形杀手，它对肌肤的伤害非常的隐蔽，没有明显的征兆，但是伤害却是不可逆的。UVA不仅能够引起黑色素沉着，让皮肤“黑化”，也会通过诱导生成ROS，造成胶原蛋白的流失，使得肌肤逐步衰老，出现皱纹、松弛和黑色暗沉等现象。因此，同时抵御黑色素沉积和真皮层细胞水平的老化是抗衰的高效途径。
L-抗坏血酸(L-Ascorbic acid)是一种天然水溶性抗氧化剂美白，又名维生素C，被广泛应用于化妆品的抗衰老成分，然而，其对光和氧敏感，不稳定的结构限制了其在化妆品工业中的应用。
有鉴于此，特提出本公开。
发明内容
本公开的目的之一在于提供一种抗坏血酸多肽衍生物，全新结构的抗坏血酸多肽衍生物，兼具了VC美白和肌肽抗衰的优异功效，并解决了传统VC结构的稳定性问题。
本公开的目的之二在于提供一种抗坏血酸多肽衍生物的制备方法。
本公开的目的之三在于提供一种抗坏血酸多肽衍生物在制备化妆品中的应用。
本公开的目的之四在于提供一种包含抗坏血酸多肽衍生物作为有效成分的化妆品。
为了实现本公开的上述目的，特采用以下技术方案：
本公开提供了一种抗坏血酸多肽衍生物，由下列通式(1)代表的化合物或其盐：
Figure PCTCN2022089635-appb-000001
其中，m为0且n为1，或者，m为1且n为0；
X 1、X 2独立的为C1～C6的烷基、C1～C6的烷氧基或卤素基；
Q 1、Q 2独立的为C1～C6的烷基链；
a、b、c独立的选自1-10的整数；
e为0且d为1-10的整数，或者，e为1且d为0。
在一些实施方式中，所述抗坏血酸多肽衍生物为由下列通式(3)代表的化合物：
Figure PCTCN2022089635-appb-000002
在一些实施方式中，所述抗坏血酸多肽衍生物为由下列通式(4)代表的化合物：
Figure PCTCN2022089635-appb-000003
在一些实施方式中，所述抗坏血酸多肽衍生物为由下列通式(6)代表的化合物：
Figure PCTCN2022089635-appb-000004
本公开提供了一种上述抗坏血酸多肽衍生物的制备方法，包括：
先对具有氨基保护基团的原料A进行羧基活化，再和原料B反应，脱保护基团后得到所述抗坏血酸多肽衍生物；
其中，所述原料A为由下列通式(7)代表的化合物：
Figure PCTCN2022089635-appb-000005
a、b独立的选自1-10的整数；
所述原料B为由下列通式(8)代表的化合物：
Figure PCTCN2022089635-appb-000006
k选自1-10的整数；
Q为C1～C6的烷基链；
Y为C1～C6的烷基、C1～C6的烷氧基或卤素基。
在一些实施方式中，所述原料A为具有氨基保护基团的肌肽；所述原料B为3-O-乙基抗坏血酸；
所述制备方法包括以下步骤：
具有氨基保护基团的原料A先与二环己基碳二亚胺、N-羟基琥珀酰亚胺在有机溶剂中反应，反应结束后除去副产物，再加入原料B和缚酸剂反应，再脱保护基团，得到所述抗坏血酸多肽衍生物。
本公开提供了一种上述抗坏血酸多肽衍生物在制备化妆品中的应用。
本公开提供了一种包含上述抗坏血酸多肽衍生物作为有效成分的化妆品；所述抗坏血酸多肽衍生物的有效浓度为10～50ppm。
本公开提供的抗坏血酸多肽衍生物至少具有如下有益效果：
本公开将抗坏血酸类衍生物结构和多肽类结构进行偶合，经理性化分子改良修饰设计合成一种新的抗坏血酸多肽衍生物，化学结构清晰明确。
全新结构的抗坏血酸多肽衍生物结构稳定，生物安全性高，解决了传统VC结构不稳定的问题。
该类抗坏血酸类衍生物经功效评价证明了其在美白、抗衰维度的显著协同性功效。细胞实验证明，化合物具有良好的生物活性，包括降低ROS，促进胶原蛋白I的产生，降低酪氨酸酶的活性和下调黑色素的分泌。在24.25μM的浓度下，EAC-L-肌肽可将黑色素的分泌下调27.85％，大于相同浓度下的EAC和肌肽相关效果的简单叠加。在皮肤弹性，皮肤色调和减少皱纹方面具有理想的抗衰老作用，是一种有前途的抗衰老活性物质，可广泛应用于化妆品、护肤品等产品中。
此外，本公开获得的相关抗坏血酸类衍生物还具有抗糖化，抗蓝光，抗自噬等功效，并可用于各部位皮肤(手，面部，四肢，头皮等)。
附图说明
为了更清楚地说明本公开实施方式或现有技术中的技术方案，下面将对实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例1的3-O-乙基抗坏血酸-肌肽的合成路线图；
图2为实施例1合成的3-O-乙基抗坏血酸-肌肽的HPLC谱图；
图3为实施例1合成的3-O-乙基抗坏血酸-肌肽的MS谱图；
图4为实施例1合成的3-O-乙基抗坏血酸-肌肽的核磁共振氢谱；
图5为实施例1合成的3-O-乙基抗坏血酸-肌肽的H，H-COSY谱；
图6为试验例1SIRC角膜细胞刺激性试验结果图；
图7为试验例1腐蚀性鸡胚绒毛尿囊膜试验结果图；
图8为试验例2酪氨酸酶酶活试验结果图；
图9为试验例2黑色素试验结果图，其中(a)为不同原料下试验结果，(b)为不同浓度下试验结果；
图10为试验例2角质形成细胞的能量代谢能力试验结果图；
图11为试验例2氧化应激下角质层细胞抗衰修复试验结果图；
图12为试验例2永生化角质形成细胞(HaCaT)预防UVA诱导损伤试验结果图，其中(a)为样品细胞毒性检测图；(b)为预防UVA损伤后的细胞活率(CSR)；(c)为预防UVA损伤后的细胞内活性氧(ROS)表达情况；(d)为预防UVA损伤后的细胞内羟脯氨酸(HYP)表达情况；
图13为试验例2成纤维细胞I型胶原蛋白的合成试验结果图；
图14为试验例2人包皮成纤维细胞(HFF)预防UVA诱导损伤试验结果图，其中(a)为预防UVA损伤后的细胞活率(CSR)；(b)为预防UVA损伤后的细胞内活性氧(ROS)表达情况；(c)为预防UVA损伤后的细胞内羟脯氨酸(HYP)表达情况；
图15为HFF细胞在UVA诱导损伤下胶原蛋白Ⅰ含量结果图；
图16为试验例3试验组人群皮肤弹性值的结果图；
图17为试验例3试验组人群ITA皮肤色泽度的结果图。
具体实施方式
下面将结合实施例对本公开的技术方案进行清楚、完整地描述，显然，所描述的实施例是本公开一部分实施例，而不是全部的实施例。基于本公开中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本公开保护的范围。
调节黑色素生成，胶原蛋白合成以及细胞再生活性是化妆品活性成分开发中的重要目标。水溶性抗氧化剂L-抗坏血酸(维生素C)超不稳定结构限制了其在化妆品工业中的应用。
为了解决这个问题，本公开开发了一种有效且稳定的多功能抗坏血酸多肽衍生物。在结构上，保留了高度还原的C＝C双键，同时，添加了多肽结构。这种新的抗坏血酸多肽不仅在细胞外基质增强，抗氧化和黑色素抑制等方面均表现出稳定的特性，而且与抗坏血酸和多肽相比抗衰老性显著改善。
根据本公开提供了一种抗坏血酸多肽衍生物，由下列通式(1)代表的化合物或其盐：
Figure PCTCN2022089635-appb-000007
抗坏血酸多肽衍生物是抗坏血酸和多肽有机(化学键)结合而形成的一种稳定的衍生物。
化合物盐一般指化合物的金属盐，例如钠盐、钾盐等。
通式(1)上述结构中：
(I)当m为0且n为1时，通式为：
Figure PCTCN2022089635-appb-000008
其中，a选自1-10的整数，a为1、2、3、4、5、6、7、8、9或10；
b选自1-10的整数，b为1、2、3、4、5、6、7、8、9或10；
e为0且d为1-10的整数(d为1、2、3、4、5、6、7、8、9或10)，或者，e为1且d为0；
Q 2为C1～C6的烷基链，可以为直链也可以为支链；
X 2为C1～C6的烷基、C1～C6的烷氧基或卤素基；
烷氧基包括但不限于甲氧基、乙氧基、乙酰氧基、异丙氧基、异丙烯氧基、叔丁氧基、叔丁基过氧基或丁酮肟；
烷基包括但不限于甲基、乙基、丙基、异丙基或丁基(正丁基、异丁基、仲丁基、叔丁基)。
卤素基包括但不限于氯基、溴基、碘基。
需要说明的是，烷基包括直链或支链的烷基。
在一些典型的实施方式中，X 2为乙氧基。
在一些典型的实施方式中，(I)的所述抗坏血酸多肽衍生物为由下列通式(2)代表的化合物或其盐：
Figure PCTCN2022089635-appb-000009
其中，R 1为C1～C6的烷基，包括但不限于甲基、乙基、丙基、异丙基或丁基(正丁基、异丁基、仲丁基、叔丁基)；烷基包括直链或支链的烷基。
a、b独立的为1-5的整数(a、b独立的为1、2、3、4或5)；
e为0且d为1-5的整数(d为1、2、3、4或5)，或者，e为1且d为0。
在一些典型的实施方式中，R 1为乙基，Q 2为C2烷基链，a为2，b为1，e为1且d为0，抗坏血酸多肽衍生物为由下列通式(3)代表的化合物：
Figure PCTCN2022089635-appb-000010
在另一些典型的实施方式中，R 1为乙基，Q 2为C1烷基链，a为2，b为1，e为0且d为1，抗坏血酸多肽衍生物为由下列通式(4)代表的化合物：
Figure PCTCN2022089635-appb-000011
通式(1)上述结构中：
(II)当m为1且n为0时，通式为：
Figure PCTCN2022089635-appb-000012
其中，a选自1-10的整数，a为1、2、3、4、5、6、7、8、9或10；
b选自1-10的整数，b为1、2、3、4、5、6、7、8、9或10；
c选自1-10的整数，c为1、2、3、4、5、6、7、8、9或10；
Q 1为C1～C6的烷基链，可以为直链也可以为支链；
X 1为C1～C6的烷基、C1～C6的烷氧基或卤素基；
烷氧基包括但不限于甲氧基、乙氧基、乙酰氧基、异丙氧基、异丙烯氧基、叔丁氧基、叔丁基过氧基或丁酮肟；
烷基包括但不限于甲基、乙基、丙基、异丙基或丁基(正丁基、异丁基、仲丁基、叔丁基)，烷基包括直链或支链的烷基。
卤素基包括但不限于氯基、溴基、碘基。
在一些典型的实施方式中，X 1为乙氧基。
在一些典型的实施方式中，(II)的所述抗坏血酸多肽衍生物为由下列通式(5)代表的化合物或其盐：
Figure PCTCN2022089635-appb-000013
其中，R 2为C1～C6的烷基,包括但不限于甲基、乙基、甲基乙基、丙基、异丙基或丁基(正丁基、异丁基、仲丁基、叔丁基)；
Q 1为C1～C6的烷基链；
a、b、c独立的为1-5的整数(a、b、d独立的为1、2、3、4或5)。
在一些典型的实施方式中，Q 1为C1烷基链，R 2为乙基，a为2，b、c独立的为1，抗坏血酸多肽衍生物为由下列通式(6)代表的化合物：
Figure PCTCN2022089635-appb-000014
本公开还提供了一种上述抗坏血酸多肽衍生物的制备方法，包括：
先对具有氨基保护基团的原料A进行羧基活化，再和原料B反应，脱保护基团后得到所述抗坏血酸多肽衍生物；
其中，原料A为由下列通式(7)代表的化合物：
Figure PCTCN2022089635-appb-000015
p选自1-10的整数,p为1、2、3、4、5、6、7、8、9或10；
q选自1-10的整数，q为1、2、3、4、5、6、7、8、9或10；
原料B为由下列通式(8)代表的化合物：
Figure PCTCN2022089635-appb-000016
k选自1-10的整数，k为1、2、3、4、5、6、7、8、9或10；
Q为C1～C6的烷基链；
Y为C1～C6的烷基、C1～C6的烷氧基或卤素基。
烷氧基包括但不限于甲氧基、乙氧基、乙酰氧基、异丙氧基、异丙烯氧基、叔丁氧基、叔丁基过氧基或丁酮肟；
烷基包括但不限于甲基、乙基、甲基乙基、丙基、异丙基或丁基(正丁基、异丁基、仲丁基、叔丁基)，烷基包括直链或支链的烷基。
卤素基包括但不限于氯基、溴基、碘基。
在一些典型的实施方式中，原料A为具有氨基保护基团的肌肽(L-Carnosine)：
Figure PCTCN2022089635-appb-000017
原料B为3-O-乙基抗坏血酸(EAC)：
Figure PCTCN2022089635-appb-000018
在一些典型的实施方式中，通式(3)、(4)、(6)代表的化合物或其盐可以按照如下反应制备：
Figure PCTCN2022089635-appb-000019
所述制备方法包括以下步骤：
具有氨基保护基团的肌肽先与二环己基碳二亚胺、N-羟基琥珀酰亚胺在有机溶剂中反应，反应结束后除去副产物，再加入3-O-乙基抗坏血酸和缚酸剂反应，再脱保护基团，得到抗坏血酸多肽衍生物。
利用二环己基碳二亚胺(DCC)和N-羟基琥珀酰亚胺(NHS)对肌肽的羧基进行活化。
有机溶剂包括但不限于DMF、二氧六环(1,4-dioxane)、丙酮。
缚酸剂包括但不限于N,N-二异丙基乙胺、二异丙基乙胺(EDIPA)、三乙胺。
反应温度和反应时间可以一般为1-30h，可选地为12-18h，反应温度可以一般为5-50℃，可选地为15-35℃。
制备方法如下：
(a)将Boc-L-Carnosine溶于DMF中，加入DCC和NHS，室温反应12-18h，过滤后出去固体副产物(N,N-二环己基脲)；
(b)在滤液中加入3-O-乙基抗坏血酸醚和DIPEA，室温反应1-5h，浓缩、干燥、重结晶，得到带有保护基团的通式(3)、(4)、(6)代表的化合物，脱保护基团后获得产物EAC-L-Carnosine。
本公开还提供了一种上述抗坏血酸多肽衍生物在制备化妆品中的应用。
本公开还提供了一种包含上述抗坏血酸多肽衍生物作为有效成分的化妆品；抗坏血酸多肽衍生物的有效浓度为10～50ppm。
本公开化合物通过将抗坏血酸类衍生物结构和多肽类结构(典型的将功效成分维生素VC(L-抗坏血酸)和肌肽(β-丙氨酰-L-组氨酸)有效结合,并进行理性化分子改良修饰设计最终开发而成。全新结构的抗坏血酸多肽衍生物，兼具了VC美白和肌肽抗衰的优异功效，并解决了传统VC结构的稳定性问题。
通过多种细胞、半体内模型以及人群功效评价来评估该创新多肽衍生物的安全性和功效性能，证明了在较低的有效浓度下(24.25μM/10ppm)可以获得良好的美白、抗衰协同功效，包括通过肌肽和L-抗坏血酸刺激胶原蛋白合成，抑制ROS和黑色素生成等。通过科学配方制备了化妆品样品，进行合理的志愿者功效测试，验证该化合物的抗衰老活性，证明了(10～50ppm)在符合经济性条件的情况下全新结构的抗坏血酸多肽衍生物可以应用于化妆品的使用场景。
下面通过实施例对本公开作进一步说明。如无特别说明，实施例中的材料为根据现有方法制备而得，或直接从市场上购得。
所用试剂：DCC，C 13H 22N 2，二环己基碳二亚胺，失水剂；NHS，N-Hydroxysuccinimide，又名HOSu，C 4H 5NO 3，N-羟基琥珀酰亚胺；DMF，二甲基甲酰胺，吸水性，水和大部分有机溶剂互溶；DIPEA，N,N-二异丙基乙胺，碱性有机溶剂；1,4-dioxane，1,4二氧六环，溶剂，稳定剂。
实施例1 3-O-乙基抗坏血酸-肌肽的合成
3-O-乙基抗坏血酸-肌肽的合成路线如图1所示，包括以下步骤：
1、首先，Boc-L-Carnosine(13g，39.9mmoL)溶于DMF(400mL)中；
2、其次，加入DCC(7.98g，38.8mmol)和HOSu(4.45g,38.8mmol)；
3、然后，溶液在室温下搅拌15h左右，过滤后出去固体副产物(N,N-二环己基脲)
4、接着，在滤液中加入3-O-乙基抗坏血酸醚(8.2g，40.2mmol)和DIPEA(16mL,92mmol)；
5、随后，混合物在室温下搅拌3h，然后浓缩，真空干燥；
6、固体用甲醇和异丙醚重结晶得到Boc-EAC-L-Carnosine(16.4g，32mmol)，为淡黄色固体；
7、Boc-EAC-L-Carnosine溶于1,4-二氧六环(100mL)中，加入4M盐酸和1,4-二氧六环(99mL，396mmol)；
8、混合物在室温下搅拌4h，然后浓缩，固体用丙酮和异丙醇研磨纯化得到EAC-L-Carnosine(3-O-乙基抗坏血酸-肌肽)(5.2g，98％)；
9、冻干后，EAC-L-Carnosine呈白色粉末(产率4.9％)。
3-O-乙基抗坏血酸-肌肽的HPLC谱图如图2所示，检测条件：泵A：100％水中0.065％三氟乙酸(v/v)；泵B：100％乙腈中0.05％三氟乙酸(v/v)；总流量：1ml/min；波长：220nm；
LC时间程序：
时间 模块 命令 值
0.01 泵 B.Conc 0
5.00 泵 B.Conc 0
20.00 泵 B.Conc 30
25.00 泵 B.Conc 65
25.01 泵 B.Conc 95
27.00 泵 B.Conc 95
27.01 泵 B.Conc 0
35.00 泵 B.Conc 0
35.01 控制器 停止 -
色谱柱：Inertsil ODS-3 4.6×250mm；
检测设备：SS-CM-0309。
出峰如下表所示：
探测器A通道1 220nm
峰号 出峰时间 面积 高度 面积比％
1 15.329 346699 40972 1.729
2 15.794 19711028 2016121 98.271
总和   20057727 2057092 100.000
3-O-乙基抗坏血酸-肌肽的MS谱图如图3所示，检测条件：接口：ESI；雾化流量：1.5L/min；CDL温度：250；块温度：200；设备：GK11010007；界面偏置：+4.5kV；干燥气体流量：5L/min；流量：0.2ml/min；B.conc：50％H 2O/50％MeOH。
3-O-乙基抗坏血酸-肌肽的核磁共振氢谱如图4所示(400MHz,溶剂D 2O)。
3-O-乙基抗坏血酸-肌肽的H，H-COSY谱如图5所示(400MHz,溶剂D 2O)。
将实施例1合成的物质作为化妆品原料，利用多种细胞模型来评估其安全性和功效，包括体外角膜细胞(SIRC)的生物安全性评估，人角质形成细胞的抗氧化活性评估(HaCaT)ROS含量降低测试，人体内胶原蛋白合成评估皮肤成纤维细胞(FB)以及酪氨酸酶活性和黑色素对黑色素瘤细胞(B16)模型的黑色素调节评估。还使用VISIA CR和CK仪器进行了抗衰老效果验证的体内研究。
试验例1安全性评估试验
实施例1合成的物质在公司自有细胞模型平台下已完成以下安全性动物替代测试，如下表：
Figure PCTCN2022089635-appb-000020
(1)SIRC角膜细胞刺激性试验：
将实施例1合成的原料配制成10ppm的溶液样品，依据中华人民共和国出入境检验检疫行业标准SN/T 3084.2-2014进行检测，结果为CSR＝106.0，表明样品为无刺激物。
检测方法：兔眼角膜细胞SIRC细胞生存率测试，将待测细胞用PBS清洗1～2次，每孔加入100μLMEM完全培 养基和10μL CCK-8溶液，置于二氧化碳培养箱中，培养2～4h。在450nm波长，测定吸光度(OD)值。CCK-8试剂可以用MTT代替。
不同样品的细胞存活率如图6所示。
(2)化妆品眼刺激性/腐蚀性鸡胚绒毛尿囊膜试验：
将实施例1合成的原料配制成10ppm的溶液样品，依据中华人民共和国出入境检验检疫行业标准SN/T 2329-2009进行检测，结果为IS＝0，表明呈阴性反应，样品无刺激性。
腐蚀性鸡胚绒毛尿囊膜试验结果如图7所示。
验证最低有效浓度下原料水溶液无任何不良反应，有很好的生物安全性。
试验例2功效评估试验
(1)美白功效
依据化妆品美白功效测试团体标准T/SHRH 021-2019，结合NCBI和知网文献调研，设计B16小鼠黑色素瘤细胞美白功效体外检测方法：
1)酪氨酸酶酶活试验：
将经过诱导剂(α-MSH)和待测物处理的细胞样品于96孔细胞培养板内，进行反复冻融裂解，每孔加入100μL的0.1～0.5％的左旋多巴，37℃细胞培养箱内孵育2h左右，在450nm波长，测定吸光度(OD)值。结果如图8所示。
2)黑色素含量测定：
将经过诱导剂(α-MSH)和待测物处理的细胞样品于6孔细胞培养板内，加入0.5～2mL细胞裂解液，在65℃恒温烘箱中，静置2h。然后，将裂解液混匀后，以150μL/孔添加到96孔检测板中，在450nm波长，测定吸光度(OD)值。结果如图9所示。
试验证明该原料可以有效抑制黑色素生成和酪氨酸酶酶活，同浓度下(24.25μM/10ppm)，相对于经典VC衍生物单体3-O-乙基抗坏血酸醚(简称EAC，一种的较为稳定的抗坏血酸衍生物)、二肽单体(肌肽)以及两者的物理混合(EAC+肌肽)相比对酪氨酸酶的抑制能力EAC-肌肽更好，而对最终黑色素的抑制能力显著提升。抑制效果与0.05％的资生堂专利成分4-MSK接近(模型的诱导剂浓度：0.1μg/mL(63nM)的α-MSH)。
可见，EAC-肌肽具有显著的美白功效，并在部分指标优于金标准成分比如3-O-乙基抗坏血酸(3-O-Ethyl Ascorbic Acid)和4-甲氧基水杨酸钾(4-MSK)等。
EAC-L-Carnosine于24.25μM(10ppm左右)存在显著抑制黑色素分泌的作用。于24.25μM浓度下，EAC-L-Carnosine的效果最优。推测可能与EAC-L-Carnosine的还原性、肌肽结构和细胞增殖有关。
(2)抗氧化抗衰老
1)角质形成细胞的能量代谢能力试验：
测试了HaCaT细胞内ATP是否会受到EAC-肌肽的影响，人永生化角质形成细胞HaCaT，将受试物处理后的细胞，使用一定量的细胞裂解液充分裂解，于4℃，12000g，离心5min，取上清，将各实验组细胞裂解样品以20μL/孔添加到96孔检测板中(不透明)，再加入100μL/孔的ATP检测液，室温下静置3～5min，使用全波段酶标仪检测自发光强度。结果如图10所示。
结果证明，相对于空白组，24.25μMEAC-肌肽能提高25.0％的ATP表达，说明其有显著性上调ATP的作用，即有提高细胞活性的作用。(注本研究中使用的实验阳性对照组1nM EGF，该原料是药品，测试浓度为1nM,不能用于护肤品)，本公开原料可显著增强角质形成细胞的能量代谢能力。
2)氧化应激下角质层细胞抗衰修复试验：
为了研究本公开原料在氧化应激下对角质层细胞抗衰修复的功效，依据化妆品紧致、抗皱功效测试-体外角质形成细胞活性氧(ROS)抑制测试方法(团体标准T/SHRH 032-2020)，通过H 2O 2诱导HaCaT细胞构建氧化损伤细胞模型，分别测定了细胞相对增值率和ROS表达量。
人永生化角质形成细胞HaCaT，铺板后，细胞融合度65％左右时，去除原有培养基，用含一定浓度的H 2O 2培养基，置于二氧化碳培养箱2h。诱导完毕后，去除原有培养基，替换为含待测样品(或阳性样品)的培养基，置于二氧化碳培养箱。
ROS检测：24h后，替换为一定量的DCFH-DA工作液，避光孵育15～30min，无血清培养基清洗3～5次后，加入100μL无血清培养基，于488nm激发波长，525发射波长，检测刺激后荧光强度。
细胞活率检测：24h后，替换为含10％的CCK-8的无血清培养基，二氧化碳培养箱孵育2～4h后，于450nm波长下检测OD值。
结果如图11所示，表示双氧水(H 2O 2)损伤后，样品修复24h后，永生化角质形成细胞(HaCaT)内ROS表达情况。
实验结果说明，该原料可以有效修复氧化损伤，显著提高细胞活力，并显著下调ROS表达量，同浓度下，与VC衍生物(EAC)、肌肽以及肌肽和EAC物理混合协同作用相比，综合功效更加优异，体现出“1+1>2”的效果。
3)UVA光老化应激下角质层细胞抗衰修复试验：
人永生化角质形成细胞HaCaT，铺板后，细胞融合度65％左右时，去除原有培养基，用含待测样品的PBS替代原有的培养基，置于特定的紫外装置下，UVA照射剂量为15J/cm 2。诱导完毕后，去除含药PBS，替换为含待测样品 的培养基，置于二氧化碳培养箱。
ROS检测：24h后，替换为一定量的DCFH-DA工作液，避光孵育15～30min，无血清培养基清洗3～5次后，加入100μL无血清培养基，于488nm激发波长，525发射波长，检测刺激后荧光强度。
细胞活率检测：24h后，替换为含10％的CCK-8的无血清培养基，二氧化碳培养箱孵育2～4h后，于450nm波长下检测OD值。
选用了一定剂量的UVA诱导损伤HaCaT，构建了角质形成细胞UVA损伤细胞模型。
图12为HaCaT细胞UVA损伤实验数据图，结论如下：
本公开EAC-肌肽(约24μM/10ppm)在无细胞毒性的情况下，有上调UVA照射后角质形成细胞的活性、显著对自由基ROS的产生有抑制效果，并对胶原蛋白前体羟脯氨酸HYP具有显著的激活作用。以上指标水平均优于VC单体(24μM)的作用，并与阳性对照TGF-β1(100ng/mL)接近。
4)成纤维细胞I型胶原蛋白的合成试验
依据化妆品、抗皱功效检测-体外成纤维细胞Ⅰ型胶原蛋白含量测定(团体标准T/SHRH 032-2020)，使用人原代成纤维细胞FB，测定了(24.25μM/10ppm)浓度下该各原料组别对FB分泌胶原蛋白的影响。
人原代成纤维细胞FB，铺板后，细胞融合度45％～65％，去除原有培养基，用含待测样品的PBS替代原有的培养基，置于二氧化碳培养箱培养24h±2h。孵育培养结束后，每孔收集200μL细胞培养上清液于1.5mL无菌离心管中，置于-80℃超低温冰箱冷冻保存。
ELISA检测，使用人Ⅰ型胶原酶免疫试剂盒。首先，使用标准品检测并绘制标准曲线，然后，检测稀释合适比例的成纤维细胞上清液含有的胶原蛋白含量。结果如图13所示。
结果证明，同浓度下，该EAC-肌肽相对于经典EAC、二肽单体以及两者的混合物有更加显著促胶原蛋白生成的效果。其中TGF-B1为阳性对照，浓度100ng/mL。可见，EAC-肌肽可显著增强成纤维细胞I型胶原蛋白的合成。
5)UVA光老化应激下对成纤维细胞抗衰修复试验
HFF人包皮成纤维细胞，铺板后，细胞融合度65％～85％时，去除原有培养基，用含待测样品的PBS替代原有的培养基，置于特定的紫外装置下，UVA照射剂量为5J/cm 2。诱导完毕后，去除含药PBS，替换为含待测样品的培养基，置于二氧化碳培养箱。
ROS检测：24h后，替换为一定量的DCFH-DA工作液，避光孵育15～30min，无血清培养基清洗3～5次后，加入100μL无血清培养基，于488nm激发波长，525发射波长，检测刺激后荧光强度。
细胞活率检测：24h后，替换为含10％的CCK-8的无血清培养基，二氧化碳培养箱孵育2～4h后，于450nm波长下检测OD值。
HYP(细胞裂解液)含量检测：使用羟脯氨酸(HYP)含量检测试剂盒。首先，使用HYP标准品检测并绘制标准曲线，然后，检测稀释合适比例的成纤维细胞裂解液中含有的HYP(细胞上清)含量。结果如图14所示。
Ⅰ型胶原蛋白含量检测：ELISA检测，使用人Ⅰ型胶原(Col Ⅰ)酶联免疫试剂盒。首先，使用Ⅰ型胶原蛋白标准品检测并绘制标准曲线，然后，检测稀释合适比例的成纤维细胞上清液含有的胶原蛋白含量。
GraphPad Prism 8.0.2软件处理，HFF细胞在UVA诱导损伤下胶原蛋白Ⅰ含量测定实验结果如图15所示。
EAC-肌肽(约24μM/10ppm)在无细胞毒性的情况下，有显著上调UVA照射后成纤维细胞的活性、显著对自由基ROS的产生有抑制效果，并对胶原蛋白前体羟脯氨酸HYP具有显著的激活作用。以上指标水平均优于VC单体/EAC单体与肌肽的混合添加协同作用，以及专利VC衍生物AA2G的作用，并与阳性对照TGF-β1接近。
可见，EAC-肌肽具有在个多维度指标上显著抗衰修护效果，并在部分指标优于经典的肌肽和VC衍生物物理混合后效果的简单叠加,具有协同的抗衰效果，实现“1+1>2”的表现。
试验例3实际志愿者人群功效
测试方法：20ppm EAC-二肽原料加入基础料乳霜中，并进行每周两次的使用，并在4周和8周进行效果的测试，以不含EAC-二肽原料的基础料乳霜为空白安慰剂。
测试人群：15人实验组，15人安慰剂组。(31-54岁,女性)。
使用方法：志愿者每天早晚使用乳霜各一次，每次使用量1g，并均匀的进行全脸涂抹。
测试设备：CK instrument。
测试方法：利用CK设备的cutometer以及Colorimeter探头分别在恒温恒湿环境下，在0周、产品使用4周和8周后对每位志愿者随机一侧脸颊进行皮肤弹性测试和皮肤色泽度测试；
空白安慰剂和测试膏霜的成分如下表所示。
Figure PCTCN2022089635-appb-000021
Figure PCTCN2022089635-appb-000022
试验组人群皮肤弹性值的结果如图16所示；结果表明：试验组人群皮肤弹性值R2，第14天和地28天有显著的提升趋势；安慰剂组提升幅度低于试验组，证明了EAC-二肽的显著应用抗衰功效。
试验组人群ITA皮肤色泽度的结果如图17所示；结果表明：试验组人群ITA皮肤色泽度，第14天和地28天有显著的提升趋势；安慰剂对照组色泽度略有下降，证明了EAC-二肽的美白提亮肤色的效果。
结论：细胞实验证明，化合物3-O-乙基抗坏血酸-肌肽具有良好的生物活性，包括降低ROS，促进胶原蛋白I的产生，降低酪氨酸酶的活性和下调黑色素的分泌。具体而言，在24.25μM的浓度下，EAC-肌肽可将黑色素的分泌下调27.85％，大于相同浓度下的EAC和肌肽的总和，证明了美白、抗衰维度的显著协同性功效，并在部分关键性指标中，该衍生物体现了同时优于VC和肌肽单体及其物理复配的功效。此外，人类志愿者的研究结果支持了3-O-乙基抗坏血酸-肌肽在皮肤弹性，皮肤色调和减少皱纹方面具有理想的抗衰老作用，具有多种功能(抗衰老、美白、抗氧化、抗炎等)，是一种有前途的抗衰老活性物质。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。

Claims (10)

  1. 一种抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(1)代表的化合物或其盐：
    Figure PCTCN2022089635-appb-100001
    其中，m为0且n为1，或者，m为1且n为0；
    X 1、X 2独立的为C1～C6的烷基、C1～C6的烷氧基或卤素基；
    Q 1、Q 2独立的为C1～C6的烷基链；
    a、b、c独立的选自1-10的整数；
    e为0且d为1-10的整数，或者，e为1且d为0。
  2. 根据权利要求1所述的抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(2)代表的化合物或其盐：
    Figure PCTCN2022089635-appb-100002
    其中，R 1为C1～C6的烷基；
    Q 2为C1～C6的烷基链；
    a、b独立的为1-5的整数；
    e为0且d为1-5的整数，或者，e为1且d为0。
  3. 根据权利要求2所述的抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(3)代表的化合物：
    Figure PCTCN2022089635-appb-100003
  4. 根据权利要求2所述的抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(4)代表的化合物：
    Figure PCTCN2022089635-appb-100004
  5. 根据权利要求1所述的抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(5)代表的化合物或其盐：
    Figure PCTCN2022089635-appb-100005
    其中，R 2为C1～C6的烷基；
    Q 1为C1～C6的烷基链；
    a、b、c独立的为1-5的整数。
  6. 根据权利要求5所述的抗坏血酸多肽衍生物，其特征在于，所述抗坏血酸多肽衍生物为由下列通式(6)代表的化合物：
    Figure PCTCN2022089635-appb-100006
  7. 一种权利要求1-6任一项所述的抗坏血酸多肽衍生物的制备方法，其特征在于，包括：
    先对具有氨基保护基团的原料A进行羧基活化，再和原料B反应，脱保护基团后得到所述抗坏血酸多肽衍生物；
    其中，所述原料A为由下列通式(7)代表的化合物：
    Figure PCTCN2022089635-appb-100007
    p、q独立的选自1-10的整数；
    所述原料B为由下列通式(8)代表的化合物：
    Figure PCTCN2022089635-appb-100008
    k选自1-10的整数；
    Q为C1～C6的烷基链；
    Y为C1～C6的烷基、C1～C6的烷氧基或卤素基。
  8. 根据权利要求7所述的制备方法，其特征在于，所述原料A为具有氨基保护基团的肌肽；所述原料B为3-O-乙基抗坏血酸；
    所述制备方法包括以下步骤：
    具有氨基保护基团的原料A先与二环己基碳二亚胺、N-羟基琥珀酰亚胺在有机溶剂中反应，反应结束后除去副产物，再加入原料B和缚酸剂反应，再脱保护基团，得到所述抗坏血酸多肽衍生物。
  9. 一种权利要求1-6任一项所述的抗坏血酸多肽衍生物在制备化妆品中的应用。
  10. 一种化妆品，其特征在于，包含权利要求1-6任一项所述的抗坏血酸多肽衍生物作为有效成分；所述抗坏血酸多肽衍生物的有效浓度为10～50ppm。
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