WO2023077338A1 - 一种六肽衍生物及其美容组合物或药用组合物和用途 - Google Patents

一种六肽衍生物及其美容组合物或药用组合物和用途 Download PDF

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WO2023077338A1
WO2023077338A1 PCT/CN2021/128560 CN2021128560W WO2023077338A1 WO 2023077338 A1 WO2023077338 A1 WO 2023077338A1 CN 2021128560 W CN2021128560 W CN 2021128560W WO 2023077338 A1 WO2023077338 A1 WO 2023077338A1
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agents
agent
optionally
synthesis
gly
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PCT/CN2021/128560
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English (en)
French (fr)
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丁文锋
彭晏
陈雪
赵文豪
孙新林
黄春青
观富宜
肖玉
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深圳市维琪科技股份有限公司
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Priority to CN202180080643.XA priority Critical patent/CN116801857A/zh
Priority to PCT/CN2021/128560 priority patent/WO2023077338A1/zh
Publication of WO2023077338A1 publication Critical patent/WO2023077338A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • 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
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • the present invention relates to a peptide derivative, a cosmetic composition or a pharmaceutical composition containing the peptide derivative and uses thereof.
  • the skin is the largest and most important organ of the human body, covering the entire surface of the human body and consisting of epidermis, dermis and subcutaneous tissue.
  • the epidermis and dermis are connected by a basement membrane, and the structural components of the basement membrane include type VII collagen, type IV collagen, laminin, integrin, intrin, and heparan sulfate glycoprotein.
  • Fibroblasts in the dermis can promote collagen synthesis, and collagen molecules cross each other to form fibers, forming a fine collagen fiber network in the skin, increasing the connection strength and stability of the skin.
  • Collagen is an important component of human skin. It has a huge water-locking function, which directly determines the hydration, smoothness, firmness and age of the skin.
  • H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 (EP1406589B1) is a collagen peptide, its structure exists simultaneously in the structure of two key basement membrane collagens IV and XVII in the human body, which can promote skin Intercellular adhesion, increase the content of type I collagen, type IV collagen, laminin-5, and integrin, promote the differentiation and maturation of epidermal cells, and promote skin regeneration, thereby exerting anti-aging effects.
  • ROS reactive oxygen species
  • the molecular structure of caffeic acid and gallic acid has phenolic hydroxyl groups, which have strong reducibility, can effectively capture oxygen free radicals, interrupt the chain reaction, thereby scavenging free radicals, protecting the body from oxidative damage, and have a wide range of uses in the fields of medicine and cosmetics .
  • Caffeic acid (Caffeic Acid, CAS No.: 331-39-5), also known as hydrolyzed coffee tannic acid, 3,4-dihydroxycinnamic acid, is a naturally occurring phenolic acid compound, commonly found in coffee drinks, apples, and blueberries and other types of food. In recent years, with the in-depth research on the biological activity of caffeic acid, it is found that it also has a good application in cosmetics. Caffeic acid has good antioxidant activity and is able to protect the skin from UV rays. Therefore, caffeic acid can be used as an anti-aging and sunscreen ingredient in cosmetics.
  • Gallic acid (Gallic Acid, CAS No.: 149-91-7), scientific name is 3,4,5-trihydroxybenzoic acid, widely exists in plants such as Rhubarb palmatum, Eucalyptus eucalyptus, Cornus officinalis, etc. A polyphenolic compound. Japan has used 95% gallic acid as an antioxidant in the 1980s. At present, gallic acid and its derivatives are widely used as free radical scavengers in the fields of biology, medicine, food, and chemical industry.
  • H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 in combination with caffeic acid and gallic acid, H-Gly-Pro-Gln-Gly-Pro-Gln- NH 2 has good solubility in water.
  • caffeic acid is slightly soluble in cold water
  • gallic acid is insoluble in cold water. Heating and dissolving may affect the stability of the polypeptide, and caffeic acid and gallic acid have certain cytotoxicity.
  • the combination The feasibility of the formulation is yet to be studied, and the effect of combined use is yet to be verified. Therefore, it is necessary to study a novel compound with high-efficiency anti-photoaging and anti-oxidation effects to overcome the defects in the prior art.
  • the inventors of the present invention have conducted experimental research to link Gly-Pro-Gln-Gly-Pro-Gln with caffeic acid or gallic acid through an amide bond to obtain the peptide derivatives of the present invention, and surprisingly found that compared to Gly- When Pro-Gln-Gly-Pro-Gln, caffeic acid or gallic acid exist alone, the anti-photoaging repair ability and anti-oxidation effect of the peptide derivative of the present invention are greatly improved, and unexpected technical effects have been achieved.
  • These peptide derivatives, cosmetic compositions or pharmaceutical compositions containing these peptide derivatives can be used to treat, prevent and/or repair signs of aging and/or photoaging of the skin, and exert antioxidant effects by scavenging free radicals , can be used in cosmetics, medicine and other fields.
  • the present invention provides a peptide derivative represented by formula (I), or its stereoisomer, or a mixture of stereoisomers,
  • R is selected from: substituent (CA-) or substituent (GA-), wherein substituent (CA-) is
  • R 2 is selected from: -NR 3 R 4 or -OR 3 , wherein each R 3 and R 4 are independently selected from each other: H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • the alkyl refers to having 1-24 carbon atoms (optionally having 1-16 carbon atoms; optionally having 1-14 carbon atoms; optionally having 1-12 carbon atoms; optionally having 1, 2 , 3, 4, 5, or 6 carbon atoms) saturated aliphatic straight-chain or branched-chain alkyl; optionally selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, Pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5- methylhexyl;
  • the alkenyl refers to having 2-24 carbon atoms (optionally having 2-16 carbon atoms; optionally having 2-14 carbon atoms; optionally having 2-12 carbon atoms; optionally having 2, 3 , 4, 5, or 6 carbon atoms) straight-chain or branched alkenyl; said alkenyl has one or more carbon-carbon double bonds, optionally with 1, 2, or 3 conjugated or non-conjugated The carbon-carbon double bond of; said alkenyl is bound to the rest of the molecule through a single bond; optionally selected from: vinyl, oleyl, or linoleyl;
  • R 3 and R 4 are independently selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;
  • R is H and R is selected from: H, methyl, ethyl, hexyl, dodecyl or hexadecyl;
  • R 2 is -OH or -NH 2 .
  • the peptide derivative represented by formula (I), or its stereoisomer, or the mixture of stereoisomers, is selected from the following peptide derivatives (1)-(4):
  • -Pro- is mentioned in the present invention, it should be understood that -Pro- is selected from -L-Pro-, -D-Pro-, or a mixture of both, and is racemic or non-racemic.
  • the preparation methods described in this document enable a person skilled in the art to obtain each stereoisomer of the peptide derivatives of the invention by selecting the amino acid with the correct configuration.
  • the synthesis of the peptide derivative represented by formula (I) of the present invention or its stereoisomer can be carried out according to conventional methods known in the prior art, such as solid-phase synthesis, liquid-phase synthesis or solid-phase and liquid Combined methods can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins with animal, fungal, or plant origin.
  • a method for obtaining a peptide derivative represented by formula (I) comprises the following steps:
  • the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising combining an amino acid having a protected N-terminus and a free C-terminus with a free N-terminus and a Amino acid coupling of the C-terminus bound to the polymer carrier; elimination of the group protecting the N-terminus; and repeating this sequence as many times as required to thereby obtain a peptide of the desired length, followed by cleavage of the desired length from the original polymer carrier Synthetic peptides.
  • the functional groups of the side chains of the amino acids remain fully protected with temporary or permanent protecting groups throughout the synthesis and can be deprotected simultaneously or orthogonally with the process of cleavage of the peptide from the polymeric support.
  • solid phase synthesis can be performed by a convergent strategy in which dipeptides or tripeptides are coupled to a polymeric support or to dipeptides or amino acids previously bound to a polymeric support.
  • the method may comprise the additional steps of deprotecting the N-terminus and C-terminus and/or cleaving the peptide from the polymer support in a non-determined order using standard conditions and methods known in the art, which may subsequently be modified. terminal functional groups.
  • the optional N-terminal and C-terminal modifications can be made to the peptide bound to the polymeric support, or after the peptide has been cleaved from the polymeric support.
  • Another aspect of the present invention provides a cosmetic or pharmaceutical composition, comprising an effective amount of the peptide derivative represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, and at least An excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.
  • the adjuvant is selected from: a collagen synthesis stimulator, an agent that regulates the synthesis of PGC-1 ⁇ , an agent that regulates the activity of PPAR ⁇ , an agent that increases or decreases the triglyceride content of adipocytes, stimulates or delays the adipocyte Differentiation agents, lipolytic agents or lipolysis stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinases Inhibitors, melanin synthesis stimulators or inhibitors, whitening or depigmenting agents, hyperpigmentation agents, self-tanning agents, antiaging agents, NO-synthase inhibitors, 5 ⁇ -reductase inhibitors, lysyl hydroxylation Enzyme and/or prolyl hydroxylase inhibitors, antioxidants, free radical scavengers and/or anti-air pollution agents,
  • the formulation of said cosmetic or pharmaceutical composition is selected from the group consisting of creams, oils, milks, balms, foams, lotions, gels, liniments, serums, soaps, shampoos, conditioners, Serum, ointment, mousse, pomade, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, syrup, elixir, Polysaccharide film, jelly or gelatin;
  • the capsules include: soft capsules, hard capsules, optionally gelatin capsules;
  • the tablet includes: sugar-coated tablet.
  • the cosmetically or pharmaceutically effective amount of the peptide derivatives of the invention to be administered, as well as their dosage, will depend on many factors, including age, the state of the patient, the severity of the condition or disease, the route and frequency of administration, and the drug to be used. Specific properties of peptide derivatives.
  • Cosmetically or pharmaceutically effective amount means a non-toxic amount of one or more peptide derivatives of the invention which is sufficient to provide the desired effect.
  • the peptide derivatives of the present invention are used in the cosmetic or pharmaceutical compositions of the present invention in cosmetically or pharmaceutically effective concentrations to obtain the desired effect; in a preferred form, relative to the total weight of the composition, at Between 0.00000001% (by weight) and 20% (by weight), preferably between 0.000001% (by weight) and 15% (by weight), more preferably between 0.0001% (by weight) and 10 % (by weight), and even more preferably between 0.0001% (by weight) and 5% (by weight).
  • a cosmetically or pharmaceutically acceptable delivery system or sustained release system in order to achieve better penetration of the active ingredient and/or to improve its pharmacokinetic and pharmacodynamic properties, It contains an effective amount of the peptide derivative represented by the above formula (I), or its stereoisomer, or a mixture of stereoisomers, or the above-mentioned cosmetic or pharmaceutical composition.
  • delivery system refers to a diluent, adjuvant, excipient or carrier with which the peptide derivatives of the invention are administered, selected from: water, oil or surfactants, including petroleum sources, animal sources, plant sources , or those of synthetic origin, such as and without limitation peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides , fatty alcohol, nonoxynol ether, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerin, digitonin and the like.
  • Diluents that can be used in the different delivery systems in which the peptide derivatives of the invention can be administered are known to those of ordinary skill in the art.
  • sustained release is used in the conventional sense to refer to a delivery system that provides a gradual release of the compound over a period of time, and preferably, but not necessarily, a relatively constant level of compound release over the entire period of time.
  • Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant liposomal vesicles, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges Cyclodextrins, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.
  • Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.
  • Sustained release systems can be prepared by methods known in the art and can be administered, for example, by topical or transdermal administration, including adhesive patches, non-adherent patches, occlusive patches, and microelectronic patch; or by systemic administration such as and without limitation, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into specific body parts, and preferably should A relatively constant amount of these peptide derivatives of the invention is released.
  • the amount of peptide derivative contained in the sustained release system will depend, for example, on the site where the composition is to be administered, the release kinetics and duration of the peptide derivative of the invention, and the condition to be treated and/or cared for, The nature of the condition and/or disease.
  • Another aspect of the present invention provides a peptide derivative represented by the above-mentioned formula (I), or its stereoisomer, or a mixture of stereoisomers, or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic Use of an aesthetically or pharmaceutically acceptable delivery system or sustained release system for the preparation of a cosmetic or pharmaceutical composition for the treatment, prevention and/or repair of signs of aging and/or photoaging of the skin.
  • Another aspect of the present invention provides a peptide derivative represented by the above-mentioned formula (I), or its stereoisomer, or a mixture of stereoisomers, or the above-mentioned cosmetic or pharmaceutical composition, or the above-mentioned cosmetic Use of an externally or pharmaceutically acceptable delivery system or sustained release system in the preparation of a cosmetic or pharmaceutical composition for scavenging free radicals and anti-oxidation.
  • the term “skin” is understood to mean the layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both inclusive. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, among others. In the present invention, the term “skin” includes the scalp.
  • treatment refers to administering the peptide derivatives according to the present invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with such a disease or condition.
  • treatment also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or disorder.
  • prevention refers to the ability of the peptide derivatives of the present invention to prevent, delay, or hinder the occurrence or development of a disease or condition before it occurs.
  • aging refers to the changes the skin undergoes as it ages (natural aging), or through exposure to sunlight (photoaging) or exposure to environmental pollutants such as chemical dirt or pollutants, tobacco smoke, etc. changes, and includes all externally visible and/or perceptible changes by touch, such as and not limited to: the development of discontinuities in the skin (such as wrinkles, fine lines, expression lines, stretch marks, striae, furrows wrinkles, unevenness or roughness, increased pore size, loss of moisture, loss of elasticity, loss of firmness, loss of smoothness, loss of ability to recover from deformation, loss of resilience), sagging of the skin (such as sagging cheeks, bags under the eyes, or double chin, etc.), changes in skin color (such as scarring, redness, bags under the eyes, or areas of hyperpigmentation such as age spots or freckles), abnormal differentiation, hyperkeratinization, elastosis, keratosis, hair loss, orange Dermoid skin, loss of collagen structure, and other histologic
  • photoaging refers to premature aging of the skin due to long-term exposure of the skin to ultraviolet radiation, which exhibits the same physiological characteristics as natural aging, such as and not limited to: sagging, sagging, color changes or hyperpigmentation Regular, abnormal and/or hyperkeratosis.
  • Gly represents NH2 - CH2 -COOH
  • Gly- represents NH2 - CH2 -CO-
  • -Gly represents -NH- CH2 -COOH
  • -Gly- represents -NH- CH2 -CO-
  • the hyphen denoting a peptide bond eliminates the OH in the 1-carboxyl group of an amino acid (represented here in conventional non-ionized form) when located to the right of the symbol, and eliminates the OH when located to the left of the symbol.
  • H in the amino acid 2-amino both modifications can be applied to the same symbol (see Table 1).
  • CA- caffeoyl
  • GA- galloyl
  • the peptide derivatives described in the present invention are obtained through artificial design and are easy to synthesize.
  • 500ppm caffeic acid has significant toxicity to fibroblasts
  • gallic acid also has significant toxicity at 100ppm and 500ppm concentrations, and by covalently binding it to the N-terminal of Gly-Pro-Gln-Gly-Pro-Gln
  • the peptide derivatives not only have no toxic effect on fibroblasts, but can also promote their proliferation.
  • the peptide derivative of the present invention is a new structural compound obtained by linking the N-terminal of Gly-Pro-Gln-Gly-Pro-Gln with caffeic acid or gallic acid through an amide bond.
  • Gly-Pro-Gln When Gly-Pro-Gln, caffeic acid or gallic acid exist alone, the anti-photoaging repair ability and anti-oxidation effect of the peptide derivative of the present invention are greatly improved, and unexpected technical effects have been achieved, showing better anti-aging Efficacy, can be used in medicine, cosmetics and other fields.
  • Figure 2 is a diagram of the staining morphology of ⁇ -galactosidase.
  • Fig. 3 is a graph showing the statistical results of ⁇ -galactosidase staining of senescent cells by peptide derivatives of the present invention at a dose of 10 ppm.
  • Fig. 4 is a graph showing the effect of the peptide derivatives of the present invention on fibroblast ROS.
  • Amide Resin a starting resin for peptide synthesis
  • Fmoc 9-fluorenylmethoxycarbonyl
  • Fmoc-linker 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl] Phenoxyacetic acid
  • DMF N,N-dimethylformamide
  • DCM dichloromethane
  • DIC diisopropylcarbodiimide
  • Ac 2 O acetic anhydride
  • DIPEA diisopropylethylamine
  • piperidine piperidine
  • HOBt 1-hydroxybenzotriazole
  • TFA trifluoroacetic acid
  • TIS triisopropylsilane
  • Gly glycine
  • Pro proline
  • Gln glutamine
  • Trt trityl Triphenylmethyl or Trityl
  • Trityl Triphenylmethyl or Trityl
  • CA (Caffeic Acid, caffeic acid);
  • CA- (caffeoyl);
  • GA (Gallic Acid, gallic acid);
  • Example 1 Preparation of R 1 -Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-Linker-Amide Resin, wherein R 1 is caffeoyl (CA-) or galloyl (GA-).
  • Fmoc-Linker-Amide Resin was de-Fmoc twice with 500ml 20% piperidine/DMF, the first reaction was 5 minutes, the second reaction was 8 minutes, and the solvent was drained for 3 minutes. Then add 100ml DMF, blow in nitrogen, start stirring for 1-2min, drain for 3min, and wash the resin repeatedly 7-8 times. Aspirate off the solvent.
  • the N-terminal Fmoc group was deprotected, and in the presence of 18.2g HOBt and 21g DIC, the activated 15.2g Fmoc-Pro-OH was coupled to the peptidyl resin using DMF as solvent, and the reaction was continued 2h. The resins were then washed and the deprotection treatment of the Fmoc group repeated for coupling of the next amino acid.
  • the sedimentation liquid is stratified and the color is yellowish white. Take out the supernatant, and pour the lower sediment into two 1L centrifuge bottles for centrifugation. Rotate at 4000rpm, centrifuge for 2min, and pour off the supernatant.
  • the solid in the centrifuge bottle was blown dry with weak nitrogen for 5 minutes. Seal it, put it in a vacuum desiccator, and vacuum it for 2 to 3 hours.
  • the crude products of CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 and GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 were obtained respectively.
  • Embodiment 3 adopts high performance liquid chromatography to carry out preparation and purification
  • Mobile phase A prepare pure acetonitrile
  • mobile phase B pure aqueous solution of 0.1% glacial acetic acid
  • the filtered sample was injected and purified, the fractions were collected, concentrated and freeze-dried to obtain the peptide CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 with a purity of 99.12%.
  • the filtered sample was injected and purified, the fractions were collected, concentrated and freeze-dried to obtain the peptide GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 with a purity of 98.69%.
  • PBS Phosphate buffered saline
  • MTT thiazolium blue
  • DMSO dimethyl sulfoxide
  • DMEM high glucose medium
  • fetal bovine serum Gibco
  • CA caffeic acid
  • GA gallic acid
  • Microplate reader (MD, USA), CO 2 incubator (Shanghai Yiheng), ultra-clean bench (Suzhou Purification).
  • the test concentration is 1ppm, 10ppm, 100ppm, 500ppm respectively;
  • the test concentration is 1ppm, 10ppm, 100ppm, 500ppm respectively;
  • test concentrations were 1ppm, 10ppm, 100ppm, 500ppm;
  • Peptide (2) CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2 , the test concentrations were 1ppm, 10ppm, 100ppm, 500ppm;
  • the tested concentrations were 1 ppm, 10 ppm, 100 ppm, and 500 ppm, respectively.
  • the purpose of this experiment is to determine the effect of the peptide derivatives of the present invention on cell proliferation by evaluating the cell activity 72 hours after administration using NIH3T3 cells as the experimental object.
  • the cell suspension was inoculated on a 96-well plate, 200 ⁇ L/well, and cultured in a constant temperature CO 2 incubator for 24 hours.
  • the medium was changed, and the samples of the administration group and the control group were added respectively, 20 ⁇ L/well, and placed in a 37° C., 5% CO 2 incubator and incubated for 72 hours.
  • MTT method is a method to detect cell survival and growth, and the measured OD value is directly proportional to cell activity.
  • Fig. 1 shows the effect of the peptide derivatives of the present invention on the proliferation of NIH3T3 cells.
  • the results show that, compared with the control group, CA has significant toxicity to NIH3T3 cells at 500ppm, and GA also has significant toxicity at 100ppm and 500ppm concentrations. Refer to Cell viability can be improved when specific peptide is in the range of 10ppm-500ppm.
  • Peptide (2) can significantly improve cell activity at 500ppm, and peptide (4) It also has no toxic effect on NIH3T3 cells at high concentrations. This shows that after CA and GA are respectively covalently combined with the N-terminus of the reference peptide to obtain the peptide derivatives of the present invention, the toxic effects of the original CA and GA on NIH3T3 cells disappear.
  • the peptide derivatives of the present invention not only have no toxic effect on fibroblasts, but can also promote their proliferation.
  • Fetal bovine serum Gibco
  • DMEM medium Gibco
  • penicillin streptomycin
  • X-gal Solarbio
  • potassium ferricyanide Solarbio
  • potassium ferrocyanide Solarbio
  • the test concentration is 1ppm, 10ppm, 100ppm respectively;
  • the test concentration is 10ppm
  • Reference peptides, test concentrations were 1ppm, 10ppm, 100ppm;
  • Peptide (2) test concentrations were 1ppm, 10ppm, 100ppm;
  • the purpose of this experiment is to use ⁇ -galactosidase staining test to test the effect of the peptide derivative of the present invention on cell aging, so as to evaluate its ability to resist photoaging.
  • cryopreserved NIH3T3 cells were cultured and subcultured at a ratio of 1:2 to about 5 passages, and the cells with better growth were selected as the experimental subjects.
  • Discard the medium add 100 ⁇ L 4% paraformaldehyde to each well, fix at room temperature for 15 min, wash with PBS three times, add ⁇ -galactosidase staining reagent (X-gal 0.01%, potassium ferricyanide 0.16%, ferrocyanide Potassium 0.02%) 50 ⁇ L/well, sealed with plastic wrap, and stained in the dark for 12 hours in a 37°C incubator. The staining solution was discarded, washed 3 times with PBS, and photographed under an optical microscope. The number of cells in the field of view and the number of stained cells were counted.
  • ⁇ -galactosidase staining reagent X-gal 0.01%, potassium ferricyanide 0.16%, ferrocyanide Potassium 0.02%
  • UV irradiation can cause cell senescence, and the cytoplasm of senescent cells will be stained blue-green by the ⁇ -galactosidase reagent (indicated by the dark part in the black and white figure).
  • the anti-photoaging ability of the peptide derivatives of the present invention was evaluated through the staining morphogram and aging cell staining statistics.
  • Fig. 2 and Fig. 3 are respectively the ⁇ -galactosidase staining morphology diagram and the ⁇ -galactosidase staining statistical result diagram of the peptide derivative of the present invention at a dose of 10 ppm to senescent cells.
  • the results showed that after UV irradiation, senescent cells increased significantly (as shown in the dark part in Figure 2); after administration, CA could not reduce the proportion of senescent cells at the test concentration, and even more senescent cells were observed at 10ppm , GA and the reference peptide could not reduce the proportion of senescent cells, indicating that neither CA, GA nor the reference peptide could improve cell photoaging.
  • peptide (2) of the present invention can significantly reduce the proportion of senescent cells at a concentration of 1ppm-100ppm, and peptide (4) can also reduce the proportion of senescent cells at a concentration of 10ppm.
  • CA the test concentration is 1ppm respectively;
  • the test concentration is 10ppm
  • test concentration is 1ppm, 10ppm respectively;
  • Another indication of aging is an increase in the level of intracellular oxidative stress, that is, an increase in ROS, and aging can be slowed down by reducing oxidative stress.
  • the purpose of this experiment is to test the effect of the peptide derivative of the present invention on the level of oxidative stress by using ROS staining experiment to evaluate its antioxidant effect.
  • cryopreserved NIH3T3 cells were cultured and subcultured at a ratio of 1:2 to about 5 passages, and the cells with better growth were selected as the experimental objects.
  • the culture medium was discarded, stained according to the instructions of the ROS probe, and the staining time was 20 min, and observed under a fluorescent microscope.
  • the active oxygen fluorescent probe is used for labeling to evaluate the antioxidant capacity of the peptide derivative of the present invention.
  • the results are shown in FIG. 4 , after UV radiation, the cell density decreased, and the expression of ROS in the cells in the same unit increased (as shown in the light-colored part in FIG. 4 ).
  • the reference peptide was at 1ppm, it failed to reduce the ROS level per unit cell; when CA was at 1ppm, it could reduce the ROS level per unit cell to a certain extent.
  • 1ppm of peptide (2) can significantly reduce the expression of ROS per unit cell and has excellent antioxidant capacity.
  • the peptide derivative of the present invention Compared with the reference peptide, CA or GA alone, the peptide derivative of the present invention has more significant anti-photoaging repair ability and anti-oxidation effect, and has achieved unexpected technical effects, showing better anti-aging Efficacy, can be used in medicine, cosmetics and other fields.
  • Dissolve allantoin and glycerin in water heat to 85°C, and keep warm for 30 minutes; dissolve PEG-7 glycerin cocoate and peptide (2) in water; mix the above solutions after cooling, and stir evenly to obtain a mixed solution; , preservatives, and essences are added to the above mixed solution in turn, and water is added to stir evenly to obtain toner.
  • Embodiment 10 contains the emulsion composition of peptide (4)
  • phase A dissolve glycerin, allantoin, polyacrylamide (and) C13-14 isoparaffin (and) laureth-7 in water, and heat to 85°C to obtain phase B; Quickly add phase B, homogenize at constant temperature for 3-5 minutes, cool; cool to below 60°C, add preservative, stir evenly, cool to below 45°C, add polypeptide solution and essence, and obtain emulsion.

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Abstract

提供一种肽衍生物及其美容组合物或药用组合物和用途，该肽衍生物具有以下通式：R 1-Gly-Pro-Gln-Gly-Pro-Gln-R 2。该肽衍生物通过咖啡酸、没食子酸分别与肽共价结合而得到，与单一的咖啡酸、没食子酸和单一多肽相比，具有优异的改善皮肤光老化和抗氧化作用，可以应用于化妆品、医药等领域。

Description

一种六肽衍生物及其美容组合物或药用组合物和用途 技术领域
本发明涉及一种肽衍生物、包含该肽衍生物的美容组合物或药用组合物及其用途。
背景技术
皮肤是人体最大和最重要的器官，被覆盖于整个人体的表面，由表皮、真皮和皮下组织构成。表皮层和真皮层之间通过基底膜连接，基底膜的结构成分中包含Ⅶ型胶原、Ⅳ型胶原、层粘连蛋白、整联蛋白、内联蛋白、硫酸肝素糖蛋白等。
真皮层的成纤维细胞能促进胶原蛋白合成，胶原蛋白分子相互交叉形成纤维，在皮肤中构成一张细密的胶原纤维网，增加皮肤的连接强度和稳定性。胶原蛋白是人体皮肤中的重要成分，其具有巨大的锁水功能，直接决定着皮肤的水润度、光滑度、紧致度和皮肤年龄。
皮肤衰老受到内源性因素和外源性因素的影响，随着年龄的增长，长时间的暴晒或紫外辐射，胶原蛋白将会大量流失，同时成纤维细胞合成的胶原蛋白也减少，这导致各种支撑皮肤的胶原肽键出现硬化、断裂，导致皮肤真皮层网状结构疏松，随即被破坏，出现塌陷，皮肤组织的保湿度和强韧性降低，皮肤随即出现干燥、皱纹、松弛等问题。
H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2(EP1406589B1)是一种胶原蛋白肽，其结构在人体的两种关键基膜胶原蛋白Ⅳ和ⅩⅦ的结构中同时存在，可以促进皮肤细胞间粘附，增加I型胶原蛋白、IV型胶原蛋白、层粘连蛋白-5、整联蛋白的含量，促进表皮细胞的分化成熟，促进皮肤再生，从而发挥抗衰功效。
除了年龄因素及紫外辐射之外，失控的氧自由基在皮肤老化过程中也扮演着重要角色。在有害刺激因素下，活性氧(reactive oxygen species,ROS)产生过多或发生代谢障碍并超过内源性抗氧化防御系统的清除能力时，ROS在体内增多并参与氧化生物大分子的形成，直接或间接氧化或损伤DNA、蛋白质和脂质，最终导致细胞的氧化损伤。ROS攻击正常细胞的细胞膜，引起细胞膜通透性增加；攻击成纤维细胞，导致胶原代谢异常，胶原合成减少，出现异常交联；弹性纤维也在氧自由基的攻击之下发生变性、卷曲，从而进一步影响真皮结构，加速皮肤老化。
咖啡酸、没食子酸的分子结构中具有酚羟基，还原性强，能有效捕获氧自由基，中断链 式反应，从而清除自由基，保护机体免受氧化损伤，在医药、化妆品领域有着广泛的用途。
咖啡酸(Caffeic Acid,CAS号：331-39-5)，又名水解咖啡鞣酸、3,4-二羟基肉桂酸，是天然存在的酚酸类化合物，普遍存在于咖啡饮品、苹果、蓝莓等各类食物中。近年来随着对咖啡酸生物活性的深入研究，发现其在化妆品中也有良好的应用。咖啡酸具有良好的抗氧化活性，能够保护皮肤免受紫外线照射。因此，咖啡酸可作为抗衰老、防晒功效成分应用到化妆品中。
没食子酸(Gallic Acid,CAS号：149-91-7)，学名为3,4,5-三羟基苯甲酸，广泛存在于掌叶大黄、大叶桉、山茱萸等植物中，是自然界存在的一种多酚类化合物。日本在20世纪80年代已经应用95％的没食子酸作为抗氧剂。目前，没食子酸及其衍生物作为自由基清除剂，在生物、医药、食品、化工等领域有广泛的应用。
为了增强抗衰效果，理论上可以考虑将H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2与咖啡酸、没食子酸组合使用，H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2在水中具有良好的溶解性，然而咖啡酸微溶于冷水，没食子酸难溶于冷水，加热溶解有可能影响多肽的稳定性，且咖啡酸、没食子酸具有一定的细胞毒性，实际上组合配方的可行性有待研究，组合使用的效果亦有待验证。因此，有必要研究一种具有高效的抗光老化及抗氧化作用的新型化合物，以克服现有技术存在的缺陷。
发明内容
本发明的发明人通过实验研究，将Gly-Pro-Gln-Gly-Pro-Gln与咖啡酸或没食子酸通过酰胺键连接，得到本发明的肽衍生物，而且出人意料地发现，相较于Gly-Pro-Gln-Gly-Pro-Gln、咖啡酸或没食子酸单独存在时，本发明的肽衍生物抗光老化修复能力及抗氧化作用均大幅提升，取得了预料不到的技术效果。这些肽衍生物、含有这些肽衍生物的美容组合物或药用组合物，可以用于治疗、预防和/或修复皮肤的老化和/或光老化的迹象，通过清除自由基，发挥抗氧化作用，可以应用于化妆品、医药等领域。
本发明提供一种式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，
R 1-Gly-Pro-Gln-Gly-Pro-Gln-R 2(I)
式(I)中，
R 1选自：取代基(CA-)或取代基(GA-)，其中取代基(CA-)是
Figure PCTCN2021128560-appb-000001
取代基(GA-)是
Figure PCTCN2021128560-appb-000002
R 2选自：-NR 3R 4或-OR 3，其中各个R 3和R 4彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C 1-C 4烷基；羟基；C 1-C 4烷氧基；氨基；C 1-C 4氨基烷基；C 1-C 4羰氧基；C 1-C 4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C 1-C 4烷基磺酰基；硫醇；C 1-C 4烷硫基；C 6-C 30芳氧基如苯氧基；-NR b(C＝NR b)NR bR c，其中R b和R c是独立地选自：H、C 1-C 4烷基、C 2-C 4烯基、C 2-C 4炔基、C 3-C 10环烷基、C 6-C 18芳基、C 7-C 17芳烷基、具有三至十元的杂环基、或氨基的保护基。
可选地，R 3、R 4彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
可选地，R 3是H并且R 4选自：H、甲基、乙基、己基、十二烷基或十六烷基；
可选地，R 2是-OH或-NH 2。
式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，选自下列肽衍生物(1)-(4)：
(1)CA-Gly-Pro-Gln-Gly-Pro-Gln-OH；
(2)CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2；
(3)GA-Gly-Pro-Gln-Gly-Pro-Gln-OH；
(4)GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2。
本发明的式(I)所示的肽衍生物可以作为立体异构体或立体异构体的混合物存在；例如，其所包含的这些氨基酸可以具有L-、D-的构型、或彼此独立地是外消旋的。因此，有可能获得同分异构混合物以及外消旋混合物或非对映混合物、或纯的非对映异构体或对映异构体，这取决于不对称碳的数量和存在什么同分异构体或同分异构混合物。本发明的式(I)所示的肽衍生物的优选的结构是纯的同分异构体，即，对映异构体或非对映异构体。
例如，当本发明所述-Pro-时，应理解-Pro-选自-L-Pro-、-D-Pro-、或两者的混合物，是外消旋的或非外消旋的。在本文件中描述的制备方法使本领域的普通技术人员能够通过选择具有正确构型的氨基酸来获得本发明的肽衍生物的每种立体异构体。
本发明式(I)所示的肽衍生物、或其立体异构体的合成可以根据现有技术中已知的常规方法来进行，例如固相合成法、液相合成法或固相与液相结合的方法，还可以通过以产生所希望的序列为目标的生物技术方法、或通过具有动物、真菌、或植物来源的蛋白质的控制水解来制备。
例如，一种获得式(I)所示的肽衍生物的方法包括以下步骤：
-将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和受保护的或与固体载体结合的C-末端的氨基酸偶联；
-消除保护N-末端的基团；
-重复该偶联顺序和消除保护N-末端的基团，直到获得所希望的肽序列；
-消除保护C-末端的基团或从该固体载体裂解。
优选地，C-末端与一种固体载体结合并且该方法是在固相上进行，包括将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和与一种聚合物载体结合的C-末端的氨基酸偶联；消除保护N-末端的基团；并且重复此顺序所需要的次数以便因此获得具有所希望的长度的肽，接着从最初的聚合物载体裂解所合成的肽。
在整个合成中这些氨基酸的侧链的官能团用临时或永久的保护基团保持充分地保护，并且可以与从该聚合物载体裂解肽的过程同时地或正交地脱保护。
可选地，固相合成可以通过将二肽或三肽偶联到聚合物支持体上或偶联到先前与聚合物支持体结合的二肽或氨基酸上的汇集策略(convergent strategy)来进行。
该方法可以包括如下另外的步骤：使用本领域已知的标准条件和方法对N-末端和C-末端脱保护和/或以非确定的次序从聚合物支持体裂解肽，随后可以修饰所述末端的官能团。可以对与聚合物支持体结合的肽进行N-末端和C-末端的任选的修饰，或在肽已从聚合物支持体裂解后进行N-末端和C-末端的任选的修饰。
本发明的另一方面，提供一种美容或药用组合物，包括有效量的上述式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，以及至少一种赋形剂和任选的美容上或药学上可接受的佐剂。
可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、有机溶剂、液体推进剂、皮肤调理剂、保湿剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素或着色剂、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、角质剥离剂、角质层分离剂、抗微生物剂、抗真菌剂、抑真菌剂、灭菌剂、抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、热休克蛋白、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、 抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗皮炎剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、粘合剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子生长因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物。
可选地，所述美容或药用组合物的制剂选自：霜剂、油、奶、香膏、泡沫、洗剂、凝胶、擦剂、浆液、皂、洗发精、润发乳、血清、软膏、摩丝、润发油、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、糖浆剂、酏剂、多糖薄膜、胶冻或明胶；
可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
可选地，所述片剂包括：糖衣片剂。
待施用的美容上或药学上有效量的本发明的肽衍生物以及它们的剂量将依赖于许多因素，包括年龄、患者的状态、病症或疾病的严重性、施用的途径和频率以及待使用的肽衍生物的具体性质。
“美容上或药学上有效量”意指无毒性的但足以提供希望的效果的本发明的一种或多种肽衍生物的量。在本发明的美容组合物或药用组合物中以获得希望的效果的美容上或药学上有效的浓度使用本发明的肽衍生物；在一个优选形式中，相对于组合物的总重量，在0.00000001％(按重量计)和20％(按重量计)之间，优选在0.000001％(按重量计)和15％(按重量计)之间、更优选在0.0001％(按重量计)和10％(按重量计)之间，并且甚至更优选在0.0001％(按重量计)和5％(按重量计)之间。
本发明的另一方面，提供一种美容上或药学上可接受的递送系统或缓释系统，以便实现有效成分的更好渗透和/或改进它的药物代谢动力学和药效动力学特性，其包含有效量的上述式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或上述的美容或药用组合物。
术语“递送系统”是指与本发明的肽衍生物一起施用的稀释剂、佐剂、赋形剂或载体， 它们选自：水、油或表面活性剂、包括石油来源、动物来源、植物来源、或合成来源的那些，例如并且不限于花生油、大豆油、矿物油、芝麻油、蓖麻油、聚山梨醇酯、脱水山梨糖醇酯、醚硫酸酯、硫酸酯、甜菜碱、葡萄糖苷、麦芽糖苷、脂肪醇、壬苯醇醚、泊洛沙姆、聚氧乙烯、聚乙二醇、右旋糖、甘油、毛地黄皂苷和类似物。本领域的普通技术人员已知在可以给予本发明的肽衍生物的不同递送系统中可以使用的稀释剂。
术语“缓释”以常规含义使用，指提供化合物在一段时间内逐渐释放的化合物的递送系统，且优选地但不是必须地，在整个时间段内具有相对恒定的化合物释放水平。
递送系统或缓释系统的实例是脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳米粒子。优选的递送系统或缓释系统是脂质体和微米乳液，更优选具有反胶束的内部结构的油包水型微米乳液。
缓释系统可以通过现有技术中已知的方法来制备，并且可以例如通过以下方式来给予：通过局部或经皮给药，包括粘附贴剂、非粘附贴剂、封闭贴剂、以及微电子贴剂；或通过全身给药例如并且不局限于，口服或胃肠外途径，包括鼻、直肠、皮下植入或注射、或直接植入或注射至特定身体部位中，并且优选地应该释放相对恒定量的本发明的这些肽衍生物。在该缓释系统中包含的肽衍生物的量将取决于例如该组合物将被给予的部位、本发明的肽衍生物的释放动力学和持续时间、以及有待治疗和/或护理的病状、病症和/或疾病的性质。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或上述美容或药用组合物，或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗、预防和/或修复皮肤的老化和/或光老化的迹象的美容组合物或药物组合物中的用途。
本发明的另一方面，提供一种上述式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或上述美容或药用组合物，或上述的美容上或药学上可接受的递送系统或缓释系统在制备用于清除自由基、抗氧化的美容组合物或药物组合物中的用途。
为了便于理解本发明，对在本发明所使用的一些术语和表述的含义说明如下：
在本发明中，术语“皮肤”应理解为是构成它的多个层，从最上层或角质层至最下层或皮下组织，两个端点都包括在内。这些层由不同类型的细胞组成，如角质形成细胞、成纤维细胞、黑色素细胞、和/或脂肪细胞等。在本发明中，术语“皮肤”包括头皮。
术语“治疗”，指的是给予根据本发明的肽衍生物以减轻或消除一种疾病或病症、或减少或消除与这种疾病或病症相关的一种或多种症状。术语“治疗”还涵盖了减轻或消除该疾病或病症的生理后果的能力。
术语“预防”，指的是本发明的肽衍生物在一种疾病或病症出现前防止、延迟、或阻碍其出现或发展的能力。
术语“老化”指的是皮肤随着年龄的增长经历的变化(自然老化)，或通过暴露于阳光(光老化)或暴露于环境污染物，如化学污垢或污染物、烟草烟雾等而经历的变化，并且包括所有外在可见的和/或通过触摸可感知的变化，例如并且不局限于：皮肤上的不连续性的发展(如皱纹、细纹、表情纹、拉伸纹、条纹、沟纹、不平整或粗糙、毛孔尺寸增大、水分损失、弹性损失、紧致性损失、平滑性损失、变形恢复能力损失、回弹性损失)、皮肤下垂(如脸颊下垂、眼睛下方出现眼袋、或出现双下巴等)、皮肤颜色的变化(如瘢痕、变红、眼袋、或出现色素过度沉着区域如老年斑或雀斑等)、异常分化、过度角质化、弹性组织变性、角化症、脱发、橘皮样皮肤、胶原结构损失，以及角质层、真皮、表皮、血管系统(例如出现蜘蛛静脉或毛细血管扩张症)或靠近皮肤的那些组织的其他组织学变化。
术语“光老化”指的是由于皮肤长期暴露于紫外线辐射而导致的皮肤过早老化，它呈现出与自然老化相同的生理特征，例如并且不局限于：松弛、下垂、颜色改变或色素沉着不规则、异常和/或过度角质化。
在本说明书中，用于氨基酸的缩写遵循IUPAC-IUB生化命名委员会(IUPAC-IUB Commission of Biochemical Nomenclature)在欧洲生物化学杂志(Eur.J.Biochem.1984,138:9-37)中所指定的规则。
因此，例如，Gly表示NH 2-CH 2-COOH，Gly-表示NH 2-CH 2-CO-，-Gly表示-NH-CH 2-COOH，并且-Gly-表示-NH-CH 2-CO-。因此，表示肽键的连字符消除了当位于该符号的右侧时的氨基酸(在此用常规非离子化形式来表示)1-羧基中的OH，并且消除了当位于该符号的左侧时的氨基酸2-氨基中的H；两种修饰可以应用于同一个符号(见表1)。
表1氨基酸残基的结构以及它们的单字母和三字母缩写符号
Figure PCTCN2021128560-appb-000003
Figure PCTCN2021128560-appb-000004
缩写“CA-”在本发明中用来表示咖啡酰基，缩写“GA-”在本发明中用来表示没食子酰基。
本发明相对于现有技术所取得的有益效果包括：
1、本发明所述的肽衍生物通过人工设计得到，合成方便。
2、500ppm咖啡酸对成纤维细胞具有显著毒性，没食子酸在100ppm及500ppm浓度下也具有显著毒性，而通过将其分别与Gly-Pro-Gln-Gly-Pro-Gln的N-末端共价结合得到本发明的肽衍生物之后，所述肽衍生物对成纤维细胞不仅没有毒性作用，而且还能促进其增殖。
3、本发明的肽衍生物是Gly-Pro-Gln-Gly-Pro-Gln的N-末端与咖啡酸或没食子酸通过酰胺键连接而得到的新结构化合物，相较于Gly-Pro-Gln-Gly-Pro-Gln、咖啡酸或没食子酸单独存在时，本发明的肽衍生物抗光老化修复能力及抗氧化作用均大幅提升，取得了预料不到的技术效果，表现出更好的抗衰功效，可以应用于医药、化妆品等领域。
附图说明
图1是本发明的肽衍生物对NIH3T3细胞增殖的影响图。*表示给药组与control组相比具有统计学差异，p<0.05(n＝3)。**表示给药组与control组相比差异显著，p<0.01(n＝3)。***表示给药组与control组相比差异极为显著，p<0.001(n＝3)。
图2是β-半乳糖苷酶染色形态图。
图3是本发明的肽衍生物在10ppm剂量下对衰老细胞的β-半乳糖苷酶染色统计结果图。###表示UV组与非辐射的control组相比，差异极为显著，p<0.001(n＝3)。*表示给药组与UV辐射后的对照组相比，具有统计学差异，p<0.05(n＝3)。**表示给药组与UV辐射后的对照组相比，差异显著，p<0.01(n＝3)。
图4是本发明的肽衍生物对成纤维细胞ROS的影响结果图。
具体实施方式
为了更好地理解本发明，下面结合实施例及附图对发明作详细的说明，然而，应当理解的是，这些实施例及附图仅用作说明目的，并且不旨在限制本发明的范围。
缩写
用于氨基酸的缩写遵循IUPAC-IUB的生物化学命名委员会在Eur J.Biochem.(1984)138：9-37和J.Chem(1989)264：633-673中指定的规则。
Amide Resin：一种多肽合成用的起始树脂；Fmoc：9-芴基甲氧羰基；Fmoc-linker：4-[(2,4-二甲氧基苯基)(Fmoc-氨基)甲基]苯氧乙酸；DMF：N,N-二甲基甲酰胺；DCM：二氯甲烷；DIC：二异丙基碳二亚胺；Ac 2O：乙酸酐；DIPEA：二异丙基乙胺；piperidine：哌啶；HOBt：1-羟基苯并三氮唑；TFA：三氟乙酸；TIS：三异丙基硅烷；Gly：甘氨酸；Pro：脯氨酸；Gln：谷氨酰胺；Trt：三苯甲基(triphenylmethyl)或三苯甲基(Trityl)；
CA：
Figure PCTCN2021128560-appb-000005
(Caffeic Acid，咖啡酸)；
“CA-”：
Figure PCTCN2021128560-appb-000006
(咖啡酰基)；
GA：
Figure PCTCN2021128560-appb-000007
(Gallic Acid，没食子酸)；
“GA-”：
Figure PCTCN2021128560-appb-000008
(没食子酰基)。
实施例1 R 1-Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-Linker-Amide Resin的制备，其中R 1是咖啡酰基(CA-)或没食子酰基(GA-)。
1.1 Fmoc-Linker-Amide Resin的制备
称取替代度为1.4的Amide Resin树脂20g，加入固相合成反应柱中。400ml DMF洗涤树脂1次，500ml DCM溶胀树脂30min，抽走溶剂。400ml DMF洗涤树脂3次。
称取Fmoc-linker 38.4g(71.1mmol)、HOBt(1.2eq)于干燥500ml广口三角瓶中。加DMF溶解后置于冰水浴中冷却2min，加DIC(1.5eq)活化3min，避免水汽。将活化后的Fmoc-linker加入溶胀后的树脂中反应2.5h，抽走反应液。用400ml DMF洗涤树脂3次，每次2min。
加入Ac 2O(5eq)、DIPEA(1eq)、DMF 400ml混合液密封3h后，用DMF 500ml洗涤树脂4次，DCM 500ml洗涤树脂2次。抽走溶剂。
1.2脱Fmoc
Fmoc-Linker-Amide Resin用500ml 20％piperidine/DMF脱Fmoc二次，第一次反应5min，第二次反应8min，抽干溶剂3min。接着加入100ml DMF，通入氮气，开启搅拌1～2min，抽干3min，重复洗涤树脂7～8次。抽走溶剂。
1.3投料反应
称取21g的Fmoc-Gln(Trt)-OH，18.2g的HOBt加入干燥圆底磨口烧瓶。加入DMF使其溶解，置于±5℃冰箱冷藏。10min后取出上述冰箱里的烧瓶后加入DIC，活化10min，避免水汽。将活化后的氨基酸加入脱保护后树脂中，通入氮气，开启搅拌，控制温度在25～35℃，反应2h，抽走反应液。加入100ml DMF，通入氮气，开启搅拌，树脂和溶剂均匀混合后计时搅拌1～2min，抽干3min。反复用100ml DMF洗涤树脂，抽干溶剂，直至抽滤出的溶剂澄清透明。
对N-末端Fmoc基团进行脱保护，并且在存在18.2g HOBt和21g DIC的情况下，使用 DMF作为溶剂，将活化后的15.2g Fmoc-Pro-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在每次偶联中，在存在18.2g HOBt和21g DIC的情况下，使用DMF作为溶剂，顺序地偶联13.4g的Fmoc-Gly-OH；27.5g的Fmoc-Gln(Trt)-OH；18.3g的Fmoc-Pro-OH；16.1g的Fmoc-Gly-OH；以及随后8.1g的CA或7.7g的GA。
在该合成之后，加入100ml DMF，通入氮气，开启搅拌，树脂和溶剂均匀混合后计时搅拌1～2min，抽干3min。反复用100ml DMF洗涤树脂，抽干溶剂，直至抽滤出的溶剂澄清透明。
1.4收缩晾干树脂
加入700ml DCM，开启搅拌，通入氮气，计时3min，抽干3min。加入200ml甲醇，开启搅拌，通入氮气，计时5min，抽干3min。再次加入200ml甲醇，开启搅拌，通入氮气，计时5min，抽干30min。将树脂倒出至托盘晾干，碾散，使树脂成流沙状，分别得到CA-Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-Linker-Amide Resin、GA-Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-Linker-Amide Resin，称重密封冷藏。
实施例2 R 1-Gly-Pro-Gln-Gly-Pro-Gln-NH 2的制备，其中R 1是咖啡酰基(CA-)或没食子酰基(GA-)。
2.1裂解
称取30g所获得的干燥的实施例1的肽基树脂，加入圆底烧瓶中。分别量取5.625ml水、213.75ml TFA、5.625ml TIS，混合均匀后加入装有肽基树脂的圆底烧瓶中，磁力搅拌，500rpm，处理2.5h。
裂解后，用砂芯漏斗抽真空过滤裂解液树脂，抽干1min。加入15ml TFA洗涤树脂，抽干1min。再次加入15ml TFA洗涤树脂，抽干1min。得到过滤后的肽裂解液。
2.2沉降
将2L冷冻后的异丙醚加入到5L血清瓶中，开启搅拌。过滤后的肽裂解液缓慢加入到5L血清瓶中，搅拌5min，静止30min。
沉降液分层，颜色黄白。抽出上清液，下层沉淀倒入2个1L离心瓶离心。转速4000rpm，离心2min，倾倒上清液。
用冷冻后的异丙醚洗涤沉淀，每1L离心瓶加入100ml异丙醚，搅拌5min，离心2min， 转速4000rpm，倾倒上清液，重复洗涤4次。
离心瓶内固体用弱氮气吹干5min。密封，放入真空干燥器，抽真空2～3h。分别得到CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2粗品、GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2粗品。
实施例3采用高效液相色谱仪进行制备纯化
3.1预处理
称取11.2g CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2粗品，用30ml乙酸，30ml甲醇和300ml水的混合溶剂溶解，经0.22μm的滤膜过滤，备用。
称取10g GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2粗品溶于5％甲醇水中，经0.22μm的滤膜过滤，备用。
3.2纯化
采用高效液相色谱仪进行制备纯化。
流动相A：制备纯乙腈；流动相B：0.1％冰乙酸的纯水溶液；
色谱柱：汉邦DAC-100；
检测波长：215nm；
CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2的洗脱梯度：
时间(min) 流速(ml/min) 流动相A(％) 流动相B(％)
0 40 5 95
15 40 10 90
35 40 30 70
40 40 35 65
41 40 70 30
50 40 70 30
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度99.12％的肽CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2。
GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2的洗脱梯度：
时间(min) 流速(ml/min) 流动相A(％) 流动相B(％)
0 15 5 95
5 15 5 95
25 15 8 92
40 15 15 85
45 15 20 80
50 15 75 25
60 15 75 25
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度98.69％的肽GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2。
实施例4
本发明式(I)中的其他化合物可以通过类似的方法制备。
所获得的这些肽通过ESI-MS测定其分子量，测试结果见下表2。
表2质谱法测定分子量
Figure PCTCN2021128560-appb-000009
Figure PCTCN2021128560-appb-000010
实施例5细胞增殖实验
5.1试剂与材料
磷酸盐缓冲液(PBS)(Gibco)、噻唑蓝(MTT)(Sigma)、二甲基亚砜(DMSO)(Sigma)、高糖培养基(DMEM)(Gibco)、胎牛血清(Gibco)、CA(咖啡酸)(Solarbio)、GA(没食子酸)(Solarbio)。
5.2仪器
酶标仪(美国MD)、CO 2培养箱(上海一恒)、超净工作台(苏州净化)。
5.3细胞株
小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。5.4待测样品
给药组：
CA，测试浓度分别为1ppm、10ppm、100ppm、500ppm；
GA，测试浓度分别为1ppm、10ppm、100ppm、500ppm；
肽H-Gly-Pro-Gln-Gly-Pro-Gln-NH 2(下称为参比肽)，测试浓度分别为1ppm、10ppm、100ppm、500ppm；
肽(2)CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2，测试浓度分别为1ppm、10ppm、100ppm、500ppm；
肽(4)GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2，测试浓度分别为1ppm、10ppm、100ppm、500ppm。
control组：
PBS空白对照。
5.5实验目的
本实验的目的是通过以NIH3T3细胞作为实验对象，评价给药后72h的细胞活性，确定本发明的肽衍生物对细胞增殖的影响。
5.6实验方法
取处于指数生长期状态良好的NIH3T3细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×10 5个/mL，制成细胞悬液。
取细胞悬液接种于96孔板上，200μL/孔，置恒温CO 2培养箱中培养24h。
换液，分别加入给药组和对照组样品，20μL/孔，置于37℃、5％CO 2培养箱中孵育72h。
之后每孔加入20μL 5mg/ml MTT，继续于37℃、5％CO 2培养箱中孵育4h。弃去原溶液，加入150μL/孔的DMSO，置于平板摇床上振摇5min后，使用酶标仪在波长570nm处测定每孔的OD值，并计算细胞活力。
细胞活力＝(给药孔OD-调零孔OD)/(对照孔OD-调零孔OD)×100％
5.7结果
MTT法是一种检测细胞存活和生长的方法，测得的OD值与细胞活性成正比。
图1是本发明的肽衍生物对NIH3T3细胞增殖的影响结果，结果显示，与control组相比，CA在500ppm时对NIH3T3细胞具有显著毒性，GA在100ppm及500ppm浓度下也具有显著毒性，参比肽在10ppm-500ppm时可以提高细胞活性。
将CA、GA分别与参比肽的N-末端通过共价键相互连接得到新结构化合物肽(2)、肽(4)，肽(2)在500ppm时能够明显提高细胞活性，肽(4)在高浓度时对NIH3T3细胞也没有毒性作用。由此表明，CA、GA分别与参比肽的N-末端共价结合得到本发明的肽衍生物之后，原CA、GA对NIH3T3细胞的毒性作用消失。本发明的肽衍生物对成纤维细胞不仅没有毒性作用，而且还能促进其增殖。
实施例6β-半乳糖苷酶染色实验
6.1试剂与材料
胎牛血清(Gibco)、DMEM培养基(Gibco)、青霉素、链霉素、X-gal(Solarbio)、铁氰化钾(Solarbio)、亚铁氰化钾(Solarbio)。
6.2仪器
CO 2培养箱、超净工作台、光学显微镜。
6.3细胞株
小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。
6.4待测样品
给药组：
CA，测试浓度分别为1ppm、10ppm、100ppm；
GA，测试浓度为10ppm；
参比肽，测试浓度分别为1ppm、10ppm、100ppm；
肽(2)，测试浓度分别为1ppm、10ppm、100ppm；
肽(4)，测试浓度为10ppm。
control组：
PBS空白对照。
6.5实验目的
本实验的目的是采用β-半乳糖苷酶染色实验测试本发明的肽衍生物对细胞衰老的影响，以评价其抗光老化的能力。
6.6实验方法
取冻存的NIH3T3细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。
将细胞以1万个/孔接种到96孔板中，待细胞长满至约80％时，建立UV光老化模型。control组加入PBS溶液，补加培养基至200μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入50μL PBS，以80J/cm 3UV灯下照射，灯源和培养瓶间距15cm，经照射后，弃去PBS，UV组加入PBS溶液和培养基至200μL，给药组加入培养基和倍比稀释药物至200μL。control组、UV组、给药组继续于37℃、5％CO 2培养箱中孵育24h。
弃去培养基，每孔加入100μL 4％多聚甲醛室温固定15min，PBS洗涤3次，加入β-半乳糖苷酶染色试剂(X-gal 0.01％，铁氰化钾0.16％，亚铁氰化钾0.02％)50μL/孔，保鲜膜封闭，37℃恒温箱避光染色12h。弃去染色液，PBS洗涤3次，置于光学显微镜下拍照。统计视野内细胞个数和染色细胞个数。
6.7结果
UV照射能够引起细胞衰老，衰老的细胞胞浆会被β-半乳糖苷酶试剂染成蓝绿色(黑白图中以深色部分表示)。本实验通过染色形态图和衰老细胞染色统计评价本发明肽衍生物的抗光老化能力。
图2、图3分别是β-半乳糖苷酶染色形态图和本发明的肽衍生物在10ppm剂量下对衰老细胞的β-半乳糖苷酶染色统计结果图。结果显示，经UV辐照后，衰老细胞明显增加(如图2中深色部分所示)；给药后，测试浓度下CA无法降低衰老细胞比例，在10ppm时甚至观察到更多的衰老细胞，GA及参比肽亦无法降低衰老细胞比例，表明CA、GA、参比肽均不能改善细胞光老化。相比之下，本发明的肽(2)在1ppm-100ppm浓度下，均能显著降低衰老细胞的比例，肽(4)在10ppm时也能够降低衰老细胞的比例。由此说明，参比肽的N-末端与CA或GA通过酰胺键连接而得到的新结构化合物，相较于参比肽、CA或GA单独存在时，表现出了预料不到的抗光老化修复能力。
实施例7 ROS染色实验
7.1试剂与材料
胎牛血清、DMEM培养基、青霉素、链霉素。
7.2仪器
CO 2培养箱、超净工作台、荧光显微镜。
7.3细胞株
小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。
7.4待测样品
给药组：
CA，测试浓度分别为1ppm；
GA，测试浓度为10ppm；
参比肽，测试浓度分别为1ppm、10ppm；
肽(2)，测试浓度分别为1ppm；
肽(4)，测试浓度为10ppm。
control组：
PBS空白对照。
7.5实验目的
衰老的另一指征是细胞内氧化应激水平提高，即ROS增加，而通过降低氧化应激可以减缓衰老。本实验的目的是采用ROS染色实验测试本发明的肽衍生物对氧化应激水平的影响，以评价其抗氧化作用。
7.6实验方法
取冻存的NIH3T3细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。
将细胞以1万个/孔接种到96孔板中，待细胞长满至约80％时，建立UV光老化模型。control组加入PBS溶液，补加培养基至200μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入50μL PBS，以80J/cm 3UV灯下照射，灯源和培养瓶间距15cm，经照射后，弃去PBS，UV组加入PBS溶液和培养基至200μL，给药组加入培养基和倍比稀释药物至200μL。control组、UV组、给药组继续于37℃、5％CO 2培养箱中孵育24h。
弃去培养基，按照ROS探针说明进行染色，染色时间为20min，并置于荧光显微镜下观察。
7.7结果
在UV光老化模型基础上，使用活性氧荧光探针进行标记，以评价本发明肽衍生物的抗氧化能力。结果如图4所示，经UV辐射后，细胞密度降低，相同单位内细胞ROS表达增加(如图4中浅色部分所示)。参比肽在1ppm时，未能降低单位细胞ROS水平；CA在1ppm时，可以在一定程度上降低单位细胞ROS水平。相比之下，1ppm的肽(2)可以显著降低单位细胞ROS的表达，具有优异的抗氧化能力。另一方面，参比肽及GA在10ppm时，均具有一定的降低ROS的作用；而10ppm的肽(4)能够更加显著地降低单位细胞ROS的表达。由此说明，参比肽的N-末端与CA或GA通过酰胺键连接而得到的新结构化合物，相较于参比肽、CA或GA单独存在时，表现出了预料不到的抗氧化作用。
相较于参比肽、CA或GA单独存在时，本发明的肽衍生物具有更加显著的抗光老化修复能力及抗氧化作用，取得了预料不到的技术效果，表现出更好的抗衰功效，可以应用于医药、化妆品等领域。
实施例8含肽(2)的爽肤水的制备
Figure PCTCN2021128560-appb-000011
将尿囊素、甘油用水溶解，加热至85℃，保温30分钟；将PEG-7甘油椰油酸酯、肽(2)用水溶解；上述溶液冷却后混合，搅拌均匀，得混合溶液；将丙二醇、防腐剂、香精依次加入上述混合溶液，加水搅拌均匀，即得爽肤水。
实施例9含肽(2)的洁面啫喱的制备
Figure PCTCN2021128560-appb-000012
将肽(2)加水溶解，搅拌均匀，得多肽溶液；将卡波姆、EDTA加水溶解，加热至85℃，保温30分钟，冷却后加入三乙醇胺，搅拌均匀，得混合溶液；将上述混合溶液、多肽溶液、防腐剂、香精依次加入水中搅拌，搅拌至完全溶胀即可得洁面啫喱。
实施例10含肽(4)的乳液组合物
Figure PCTCN2021128560-appb-000013
将0.01g肽(4)配置为0.02mg/mL的水溶液；将鲸蜡硬脂醇(和)鲸蜡硬脂基葡糖苷、霍霍巴油、矿物油、棕榈酸异丙酯加热至85℃，搅拌均匀；得A相；将甘油、尿囊素、聚丙烯酰胺(和)C13-14异链烷烃(和)月桂醇聚醚-7用水溶解，加热至85℃，得B相；将A相快速加入B相，恒温均质3-5min，冷却；冷却至60℃以下加入防腐剂，搅拌均匀，冷却至45℃以下加入多肽溶液、香精，即得乳液。
以上内容是结合具体的优选实施方式对本发明所做的进一步详细的说明，但是不表示本发明的具体实施是局限于这些说明。对于本发明所属领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或是替换，都应视为属于本发明的保护范围。

Claims (8)

  1. 式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，
    R 1-Gly-Pro-Gln-Gly-Pro-Gln-R 2 (I)
    式(I)中，
    R 1选自：取代基(CA-)或取代基(GA-)，其中取代基(CA-)是
    Figure PCTCN2021128560-appb-100001
    取代基(GA-)是
    Figure PCTCN2021128560-appb-100002
    R 2选自：-NR 3R 4或-OR 3，其中各个R 3和R 4彼此独立地选自：H、取代的或未取代的烷基、取代的或未取代的烯基；
    所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
    所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
    可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C 1-C 4烷基；羟基；C 1-C 4烷氧基；氨基；C 1-C 4氨基烷基；C 1-C 4羰氧基；C 1-C 4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C 1-C 4烷基磺酰基；硫醇；C 1-C 4烷硫基；C 6-C 30芳氧基如 苯氧基；-NR b(C＝NR b)NR bR c，其中R b和R c是独立地选自：H、C 1-C 4烷基、C 2-C 4烯基、C 2-C 4炔基、C 3-C 10环烷基、C 6-C 18芳基、C 7-C 17芳烷基、具有三至十元的杂环基、或氨基的保护基。
  2. 根据权利要求1所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，其特征在于，R 3、R 4彼此独立地选自：H、甲基、乙基、己基、十二烷基或十六烷基；
    可选地，R 3是H并且R 4选自：H、甲基、乙基、己基、十二烷基或十六烷基；
    可选地，R 2是-OH或-NH 2。
  3. 根据权利要求1-2任一项所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，其特征在于，选自下列肽衍生物(1)-(4)：
    (1)CA-Gly-Pro-Gln-Gly-Pro-Gln-OH；
    (2)CA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2；
    (3)GA-Gly-Pro-Gln-Gly-Pro-Gln-OH；
    (4)GA-Gly-Pro-Gln-Gly-Pro-Gln-NH 2。
  4. 一种美容或药用组合物，其特征在于，包括有效量的权利要求1-3任一项所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，以及至少一种赋形剂和任选的美容上或药学上可接受的佐剂；
    可选地，所述佐剂选自：胶原合成刺激剂、调节PGC-1α合成的剂、调节PPARγ的活性的剂、增加或减少脂肪细胞的甘油三酸酯含量的剂、刺激或延迟脂肪细胞分化的剂、脂解剂或刺激脂肪分解的剂、溶脂剂、生脂剂、乙酰胆碱受体聚集的抑制剂、抑制肌肉收缩的剂、抗胆碱能试剂、弹性蛋白酶抑制剂、基质金属蛋白酶抑制剂、黑色素合成刺激或抑制剂、增白剂或脱色剂、促色素沉着剂、自晒黑剂、抗老化剂、NO-合酶抑制剂、5α-还原酶抑制剂、赖氨酰羟化酶和/或脯氨酰羟化酶的抑制剂、抗氧化剂、自由基清除剂和/或抗大气污染的剂、活性羰基类物质清除剂、抗糖化剂、抗组胺剂、抗病毒剂、抗寄生虫剂、乳化剂、润肤剂、有机溶剂、液体推进剂、皮肤调理剂、保湿剂、保留水分的物质、α羟基酸、β羟基酸、增湿剂、表皮水解酶、维生素、氨基酸、蛋白质、色素或着色剂、染料、生物聚合物、胶凝聚合物、增稠剂、表面活性剂、软化剂、粘合剂、防腐剂、抗皱剂、能够减少或治疗下眼袋的剂、去角质剂、角质剥离剂、角质层分离剂、抗微生物剂、抗真菌剂、抑真菌剂、灭菌剂、 抑菌剂、刺激真皮或表皮大分子的合成和/或能够抑制或预防它们的降解的剂、刺激弹性蛋白合成的剂、刺激核心蛋白聚糖合成的剂、刺激层粘连蛋白合成的剂、刺激防御素合成的剂、刺激伴侣蛋白合成的剂、刺激cAMP合成的剂、热休克蛋白、刺激HSP70合成的剂、刺激热休克蛋白合成的剂、刺激透明质酸合成的剂、刺激纤连蛋白合成的剂、刺激去乙酰化酶合成的剂、刺激脂质和角质层组分的合成的剂、神经酰胺、脂肪酸、抑制胶原降解的剂、抑制弹性蛋白降解的剂、抑制丝氨酸蛋白酶的剂、刺激成纤维细胞增殖的剂、刺激角质形成细胞增殖的剂、刺激脂肪细胞增殖的剂、刺激黑色素细胞增殖的剂、刺激角质形成细胞分化的剂、抑制乙酰胆碱酯酶的剂、皮肤松弛剂、刺激糖胺聚糖合成的剂、抗角化过度剂、粉刺溶解剂、抗银屑病剂、抗皮炎剂、抗湿疹剂、DNA修复剂、DNA防护剂、稳定剂、止痒剂、用于治疗和/或护理敏感性皮肤的剂、固化剂、紧致剂、重构剂、抗拉伸纹剂、粘合剂、调节皮脂产生的剂、止汗剂、刺激愈合的剂、协助愈合的剂、刺激再上皮化的剂、协助再上皮化的剂、细胞因子生长因子、镇静剂、抗炎剂、麻醉剂、作用于毛细血管循环和/或微循环的剂、刺激血管生成的剂、抑制血管渗透性的剂、静脉紧张剂、作用于细胞代谢的剂、用于改善真皮-表皮接合的剂、诱导毛发生长的剂、毛发生长抑制或延缓剂、香料、螯合剂、植物提取物、精油、海洋提取物、得自生物发酵过程的剂、无机盐、细胞提取物、防晒剂、以及有效抗A和/或B紫外线的有机或无机光防护剂或其混合物。
  5. 根据权利要求4所述的美容或药用组合物，其特征在于，所述美容或药用组合物的制剂选自：霜剂、油、奶、香膏、泡沫、洗剂、凝胶、擦剂、浆液、皂、洗发精、润发乳、血清、软膏、摩丝、润发油、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、糖浆剂、酏剂、多糖薄膜、胶冻或明胶；
    可选地，所述胶囊剂包括：软胶囊剂、硬胶囊剂，可选为明胶胶囊剂；
    可选地，所述片剂包括：糖衣片剂。
  6. 一种美容上或药学上可接受的递送系统或缓释系统，其特征在于，包含有效量的权利要求1-3任一项所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或权利要求4或5所述的美容或药用组合物；
    所述美容上或药学上可接受的递送系统或缓释系统选自：脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳 液、毫米粒子、微米粒子或纳米粒子；可选为脂质体或微米乳液，可选具有反胶束的内部结构的油包水型微米乳液。
  7. 权利要求1-3任一项所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或权利要求4或5所述美容或药用组合物，或权利要求6所述的美容上或药学上可接受的递送系统或缓释系统在制备用于治疗、预防和/或修复皮肤的老化和/或光老化的迹象的美容组合物或药物组合物中的用途。
  8. 权利要求1-3任一项所述的式(I)所示的肽衍生物，或其立体异构体、或立体异构体的混合物，或权利要求4或5所述美容或药用组合物，或权利要求6所述的美容上或药学上可接受的递送系统或缓释系统在制备用于清除自由基、抗氧化的美容组合物或药物组合物中的用途。
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