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 PDFInfo
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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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- 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
Description
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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 | - |
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 |
Claims (10)
- 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。
- 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。
- 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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