CN117357426A - Novel use of pentapeptides - Google Patents

Novel use of pentapeptides Download PDF

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Publication number
CN117357426A
CN117357426A CN202311667359.XA CN202311667359A CN117357426A CN 117357426 A CN117357426 A CN 117357426A CN 202311667359 A CN202311667359 A CN 202311667359A CN 117357426 A CN117357426 A CN 117357426A
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China
Prior art keywords
acid
peptide
formula
salt
skin
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CN202311667359.XA
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Chinese (zh)
Inventor
丁文锋
赵文豪
观富宜
肖玉
孙新林
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Shenzhen Weiqi Technology Co ltd
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Shenzhen Weiqi Technology Co ltd
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Priority to CN202311667359.XA priority Critical patent/CN117357426A/en
Publication of CN117357426A publication Critical patent/CN117357426A/en
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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
    • 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

Abstract

The invention discloses a new application of pentapeptides, wherein the peptides or salts thereof have a structure shown in a formula (I): r is R 1 ‑Lys‑Val‑Lys‑Leu‑Tyr‑R 2 (I) A. The invention relates to a method for producing a fibre-reinforced plastic composite In particular to the use of the peptide or salt thereof in the preparation of a composition for caring skin or mucous membrane, including one or more of restoring function of skin or mucous membrane, promoting regeneration or healing of skin or mucous membrane, improving adhesion capacity of cells, promoting collagen production, increasing skin elasticity or improving skin firmness.

Description

Novel use of pentapeptides
Technical Field
The invention belongs to the technical field of polypeptides, and particularly relates to a novel application of pentapeptides.
Background
Skin is the largest organ of human body and the most exposed part in the environment, and with the increase of age, the metabolism of skin is slowed down by various factors such as internal and external environment, collagen is continuously lost, and the elastic net supporting the skin is broken, skin tissue is atrophic and collapsed, loses luster and elasticity, and the water in dermis is lost, so that skin aging signs such as dryness, roughness, relaxation and the like appear.
Today, the desire to have a tight, smooth, elastic skin is becoming a great desire for many people, and therefore, how to effectively solve or improve the above-mentioned skin problems has become a focus of attention. It is known that collagen is an important substance constituting a skin tissue structure, and has functions of supporting and nourishing skin, imparting elasticity and firmness to skin, maintaining moisture balance of skin, promoting growth and repair of skin cells, and maintaining morphological structure of skin, so that it can supplement collagen in time, increase collagen content in vivo, and has excellent effects of improving the above-mentioned skin problems, and delaying aging. In addition, it has been shown that the adhesion of keratinocytes at the dermis-epidermis junction of the skin also plays an important role in delaying skin aging, and by increasing cell adhesion and cell-extracellular matrix adhesion between them, skin elasticity can be increased, thereby preventing skin sagging.
Disclosure of Invention
The present invention aims to provide a use of a pentapeptide or a salt thereof for preparing a composition for caring skin or mucous membrane, wherein the caring skin or mucous membrane comprises one or more of restoring the function of the skin or mucous membrane, promoting the regeneration or healing of the skin or mucous membrane, improving the adhesion capability of cells, promoting collagen production, increasing the elasticity of the skin or improving the firmness of the skin.
The peptide or a salt thereof of the present invention has the following formula (I),
R 1 -Lys-Val-Lys-Leu-Tyr-R 2 （I）
in the formula (I) of the present invention,
R 1 selected from: h or R 3 -CO-, wherein R 3 Selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
R 2 selected from: -NR 4 R 5 OR-OR 4 Wherein each R is 4 And R is 5 Independently of each other selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
alkyl refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1, 2, 3, 4, 5, or 6 carbon atoms); 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 group refers to a straight or branched alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); the alkenyl group has one or more carbon-carbon double bonds, optionally 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bound to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;
optionally, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C 1 -C 4 An alkyl group; a hydroxyl group; c (C) 1 -C 4 An alkoxy group; an amino group; c (C) 1 -C 4 An aminoalkyl group; c (C) 1 -C 4 A carbonyloxy group; c (C) 1 -C 4 An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); cyano group; a nitro group; an azide; c (C) 1 -C 4 An alkylsulfonyl group; a mercaptan; c (C) 1 -C 4 Alkylthio; c (C) 6 -C 30 Aryloxy groups such as phenoxy; -NR b (C=NR b )NR b R c Wherein R is b And R is c Is independently selected from: H. c (C) 1 -C 4 Alkyl, C 2 -C 4 Alkenyl, C 2 -C 4 Alkynyl, C 3 -C 10 Cycloalkyl, C 6 -C 18 Aryl, C 7 -C 17 Aralkyl groups, heterocyclic groups having three to ten members, or protecting groups for amino groups.
Alternatively, R 1 Selected from: H. acetyl, t-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, and nutmegAcyl, palmitoyl, stearoyl, oleoyl or linoleoyl; r is R 4 、R 5 Independently of each other selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
alternatively, R 1 Selected from H, acetyl, lauroyl, myristoyl or palmitoyl; r is R 4 Is H and R 5 Selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;
alternatively, R 1 Is H or palmitoyl; r is R 2 is-OH or-NH 2 。
Alternatively, the peptide is selected from peptides (1) - (4):
（1）H-Lys-Val-Lys-Leu-Tyr-NH 2 ；
（2）H-Lys-Val-Lys-Leu-Tyr-OH；
（3）Palm-Lys-Val-Lys-Leu-Tyr-NH 2 ；
（4）Palm-Lys-Val-Lys-Leu-Tyr-OH。
alternatively, the peptide is selected from peptide (3) or peptide (4):
（3）Palm-Lys-Val-Lys-Leu-Tyr-NH 2 ；
（4）Palm-Lys-Val-Lys-Leu-Tyr-OH。
the peptide of formula (I) of the present invention may exist as stereoisomers or as mixtures of stereoisomers; for example, the amino acids they comprise may have the L-, D-configuration, or be racemic independently of each other. Thus, it is possible to obtain isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomers or isomeric mixtures are present. The preferred structure of the peptides of formula (I) of the present invention is the pure isomer, i.e., enantiomer or diastereomer. Naturally occurring L-isomers may be preferred.
The invention also includes all suitable isotopic variants of the peptides of formula (I). Isotopic variations of these peptides of the present invention are understood herein to mean those compounds: wherein within the peptides of the inventionAt least one atom is replaced with another atom of the same atomic number, but the atomic mass of the other atom is different from the atomic mass normally or mainly present in nature. Examples of isotopes that can be incorporated into the peptides of the invention are: those of hydrogen, carbon, nitrogen or oxygen, e.g. 2 H (deuterium), 3 H (tritium), 13 C、 14 C、 15 N、 17 O or 18 O. Specific isotopic variations of the peptides of the present invention (particularly those into which one or more radioisotopes have been incorporated) may be advantageous, for example, for examining the mechanism of action or distribution of active compounds in vivo; due to relatively simple producibility and detectability, especially with 3 H or 14 C isotopically labeled compounds are suitable for this purpose. In addition, due to the greater metabolic stability of the compounds, the incorporation of isotopes (e.g., deuterium) may yield particular therapeutic benefits, such as increased in vivo half-life or reduced amounts of active agent required; thus, in some cases, such modifications of the peptides of the invention may also constitute preferred embodiments of the invention. Isotopic variants of the peptides of the invention can be prepared by methods known to those skilled in the art, for example, by methods further described below and in the examples, by using the respective reagents and/or corresponding isotopic modifications of the starting materials.
Optionally, the composition comprises a peptide of formula (I) or a salt thereof at a concentration of 0.0001% to 5% by mass;
or, the composition comprises a peptide shown in a formula (I) or a salt thereof with a mass percentage concentration of 0.0005% -1%;
or, the composition comprises the peptide shown in the formula (I) or the salt thereof with the mass percentage concentration of 0.001% -0.1%;
or the composition comprises the peptide shown in the formula (I) or the salt thereof with the mass percentage concentration of 0.005-0.01%.
The term "salt" refers to a salt approved for use in animals, and more specifically in humans, including metal salts of peptides of formula (I), including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.; the salt of the peptide of formula (I) includes a salt of the peptide of formula (I) with an organic base including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine and the like; the salt of the peptide represented by formula (I) includes a salt of the peptide represented by formula (I) with an inorganic acid or an organic acid including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate (pamoate), gluconic acid, or the like; the inorganic acids include, but are not limited to: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.
The synthesis of the peptide of formula (I) or a salt thereof of the present invention can be carried out according to conventional methods known in the art, such as a solid phase synthesis method, a liquid phase synthesis method or a method of combining a solid phase with a liquid phase, and can also be prepared by a biotechnological method aimed at producing a desired sequence, or by controlled hydrolysis of a protein having animal, fungal, or plant origin.
For example, a method for obtaining a peptide of formula (I) comprises the steps of:
-coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a protected or solid carrier-bound C-terminus;
-elimination of the group protecting the N-terminal end;
-repeating the coupling sequence and elimination of the N-terminal protecting group until the desired peptide sequence is obtained;
-elimination of the C-terminal protecting group or cleavage from the solid support.
Preferably, the C-terminus is bound to a solid support and the method is performed on a solid phase, comprising coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus bound to a polymeric support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptide of the desired length, followed by cleavage of the synthesized peptide from the original polymer carrier.
Alternatively, solid phase synthesis may be performed by a pooling strategy (convergent strategy) of coupling a dipeptide or tripeptide to a polymeric support or to a dipeptide or amino acid previously bound to a polymeric support.
The functional groups of the ends may be subsequently modified using standard conditions and methods known in the art to deprotect the N-and C-termini and/or cleave the peptide from the polymeric support in a non-defined order. The peptide of formula (I) bound to the polymeric support may be optionally modified at the N-and C-termini, or after the peptide has been cleaved from the polymeric support.
The peptide of formula (I) or a salt thereof may be incorporated into a delivery system or a sustained release system in order to achieve better penetration of the active ingredient, and the peptide of formula (I) or a salt thereof may be applied to the skin and/or mucous membrane.
The frequency of administration may vary widely depending on the needs of each subject, with recommended administration ranging from 1 per month to 10 times per day, preferably from 1 per week to 4 times per day, more preferably from 3 per week to 3 times per day, even more preferably 1 or 2 times per day.
The term "delivery system" refers to a diluent, adjuvant, excipient or carrier with which the peptides of the invention are administered, selected from the group consisting of: water, oils or surfactants, including those of petroleum origin, animal origin, vegetable origin, or synthetic origin, such as, and not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycols, dextrose, glycerol, digitonin, and the like. Diluents which can be used in different delivery systems to which the peptides of the invention can be administered are known to those of ordinary skill in the art.
The term "sustained release" is used in a conventional sense to refer to a delivery system that provides a gradual release of a compound over a period of time, and preferably, but not necessarily, has a relatively constant level of release of the compound over the entire period of time.
Examples of delivery systems or sustained release systems include, but are not limited to: liposomes, oleosomes, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, clathrates, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles.
The peptide represented by the above formula (I) or a salt thereof may be adsorbed on a solid organic polymer or a solid inorganic support, for example, and not limited to, talc, bentonite, silica, starch, maltodextrin, or the like.
Optionally, the formulation of the composition comprises: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, solution, suspension, emulsion, elixir, polysaccharide film, or jelly.
Optionally, the composition further comprises at least one active substance.
Optionally, the active substance comprises peptides, natural plant ingredients, vitamin C derivatives or retinoids.
The peptides of the invention have variable solubility in water depending on the nature of their sequences or any possible modification in the N-terminal and/or C-terminal. The peptides of the invention may thus be incorporated into the composition by aqueous solutions, and those that are insoluble in water may be dissolved in conventional solvents such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol or polyethylene glycol or any combination thereof.
In the present specification, abbreviations for amino acids follow the rules specified in the European journal of biochemistry (Eur. J. Biochem. 1984, 138:9-37) by the IUPAC-IUB Biochemical nomenclature Commission (IUPAC-IUB Commission of Biochemical Nomenclature).
Thus, for example, lys represents NH 2 -CH(CH 2 CH 2 CH 2 CH 2 NH 2 ) -COOH, lys-represents NH 2 -CH(CH 2 CH 2 CH 2 CH 2 NH 2 ) -CO-, -Lys represents-NH-CH (CH) 2 CH 2 CH 2 CH 2 NH 2 ) -COOH, and-Lys-represents-NH-CH (CH) 2 CH 2 CH 2 CH 2 NH 2 ) -CO-. Thus, the hyphen representing the peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented here in conventional non-ionized form) when located to the right of the symbol, and eliminates the H in the 2-amino group of the amino acid when located to the left of the symbol; both modifications can be applied to the same symbol (see table 1).
TABLE 1 Structure of amino acid residues and their single and three letter abbreviations
The abbreviation "Palm-" is used in the present invention to denote palmitoyl; lys: lysine; val: valine; leu: leucine; tyr: tyrosine.
The invention has the following advantages and effects:
1. the peptide is obtained through artificial design, is convenient to synthesize, and is safe and non-irritating to human bodies.
2. The peptide provided by the invention can restore the function of skin or mucous membrane, promote the regeneration or healing of the skin or mucous membrane, improve the adhesion capability of cells, promote the generation of collagen, increase the skin elasticity and improve the skin firmness, and can be widely applied to products for caring the skin or mucous membrane.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the description of the present invention will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a peptide (3) Palm-Lys-Val-Lys-Leu-Tyr-NH 2 Is a mass spectrum of (3).
FIG. 2 is a mass spectrum of peptide (4) Palm-Lys-Val-Lys-Leu-Tyr-OH.
FIG. 3 is a graph showing the effect of the test sample on the content of collagen III.
FIG. 4 is a graph showing the effect of the test sample on the collagen I content.
FIG. 5 is a graph of the effect of test samples on HaCaT cell adhesion.
Detailed Description
In order that the described objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, all reagents and materials used in the present invention are commercially available. The following are abbreviations for part of the reagents and materials:
amide Resin: a starting resin for polypeptide synthesis (crosslinking degree 1%, substitution degree 1.72mmol/g, granularity 100-200 meshes); fmoc-Linker:4- [ (2, 4-Dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetic acid; wang Resin: king resin; ac (Ac) 2 O: acetic anhydride; DMF: n, N-dimethylformamide; DIPEA: diisopropylethylamine; DIC: diisopropylcarbodiimide; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; TIS: triisopropylsilane; DMAP: 4-dimethylaminopyridine; palm-OH: palmitic acid; palm-: palmitoyl; lys: lysine; val: valine; leu: leucine; tyr: tyrosine; fmoc: 9-fluorenylmethoxycarbonyl; boc: a tert-butoxycarbonyl group; tBu: and (3) tert-butyl.
Example 1 Palm-Lys-Val-Lys-Leu-Tyr-NH 2 Is prepared from
1.1 Preparation of Fmoc-Linker-Amide Resin
9g of Amide Resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the Resin and the solvent was removed.
8.7g Fmoc-Linker, 2.6g HOBt were weighed into a drying flask. After being dissolved by DMF solvent, the mixture is placed in ice water bath for cooling for 10min, 3.7mL of DIC is added for activation for 10min, and water vapor is avoided.
Adding the activated Fmoc-Linker into the swelled resin, reacting for 2.5 hours, pumping out the reaction liquid, washing the resin, and pumping out the solvent.
Continuing to add Ac 2 O and DIPEA were capped for 1.5h. The resin was washed and the solvent was pumped away.
1.2 Fmoc removal
Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled K for detection, and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed.
1.3 Feeding reaction
7.7g of Fmoc-Tyr (tBu) -OH and 2.7g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a-18℃refrigerator for 30min. 3.9mL DIC was added to activate for 3min to avoid water vapor. And adding the activated amino acid into the deprotected resin for reaction for 2 hours, and pumping away the reaction solution. K detection resin is colorless and transparent, which indicates that the reaction is complete.
Deprotection of the N-terminal Fmoc group was performed and 5.9g of activated Fmoc-Leu-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 2.7g HOBt and 3.9mL DIC for 2h. These resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 7.9g of Fmoc-Lys (Boc) -OH, 6.8g of Fmoc-Val-OH and then 9.4g of Fmoc-Lys (Boc) -OH were coupled sequentially using DMF as solvent in the presence of 2.7g of HOBt and 3.9mL of DIC; after the reaction was completed, the resin was washed and the solvent was removed.
The N-terminal Fmoc group of the peptidyl resin was deprotected and Fmoc was removed twice with 20% pipeidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
8.6g of Palm-OH was coupled to the peptidyl Resin using DMF as solvent in the presence of 5.4g HOBt and 7.7mL DIC, the reaction was continued for 2.5h, the Resin was washed, the solvent was removed, and after shrink drying 19.8g of Palm-Lys (Boc) -Val-Lys (Boc) -Leu-Tyr (tBu) -Linker-Amide Resin was obtained.
1.4 Cleavage of
Measuring 152mL of TFA, 4mL of TIS and 4mL of water, mixing and stirring uniformly to obtain a lysate, sealing and placing in a refrigerator at the temperature of minus 18 ℃ for later use; the isopropyl ether is placed in a refrigerator at the temperature of minus 18 ℃ for refrigeration for standby.
19.8g of Palm-Lys (Boc) -Val-Lys (Boc) -Leu-Tyr (tBu) -Linker-Amide Resin was weighed, added to a round bottom flask, the frozen lysate was added, and the reaction was stirred for 2h. Suction filtration, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times, and vacuum drying to obtain 6.3g Palm-Lys-Val-Lys-Leu-Tyr-NH 2 Crude peptide.
1.5 Purification
6.3g of the crude peptide was weighed and dissolved in 450mL of a mixed solution of acetic acid, methanol and water, and the solution was filtered through a microporous membrane having a pore size of 0.22 μm to obtain a clear and transparent solution, which was purified by reverse phase HPLC, the purification gradient was as shown in the following Table:
time (min) Flow rate (mL/min) A% (acetonitrile) B% (0.1% acetic acid + pure water)
0 40 10 90
10 40 25 75
20 40 35 65
30 40 40 60
50 40 50 50
80 40 60 40
Purifying the filtered sample, collecting the fraction, concentrating and freeze-drying to obtain peptide (3) Palm-Lys-Val-Lys-Leu-Tyr-NH with purity of 96.55% 2 。
Example 2 preparation of Palm-Lys-Val-Lys-Leu-Tyr-OH
2.1 Swelling and reaction of resins
10g of Wang Resin was weighed into a solid phase synthesis reaction column, swollen with DMF, the Resin was washed, and the solvent was removed.
11.0g of Fmoc-Tyr (tBu) -OH, 4.0g of HOBt were weighed into a dry flask. Dissolving with DMF solvent, cooling in ice water bath for 10min, and adding 6mL DIC for 10min to avoid water vapor.
The activated Fmoc-Tyr (tBu) -OH and 1.5g DMAP were added to the swollen resin and reacted for 2.5h, the reaction solution was removed, the resin was washed, and the solvent was removed.
Continuing to add Ac 2 O, DIPEA and DMAP capping treatments 1.5h. The resin was washed and the solvent was pumped away.
2.2 Fmoc removal
Fmoc-Tyr (tBu) -Wang Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled K and developed dark blue. The resin was washed 7 times with DMF and the solvent was removed.
2.3 Feeding reaction
7g of Fmoc-Leu-OH and 3g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. Adding 5mL DIC to activate for 3min to avoid water vapor. And adding the activated amino acid into the deprotected resin for reaction for 2 hours, and pumping away the reaction solution. K detection resin is colorless and transparent, which indicates that the reaction is complete.
The N-terminal Fmoc group was deprotected and 9g of Fmoc-Lys (Boc) -OH after activation was coupled to the peptidyl resin using DMF as solvent in the presence of 3g HOBt and 5mL DIC for 2h. These resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 8g of Fmoc-Val-OH followed by 11g of Fmoc-Lys (Boc) -OH were coupled sequentially using DMF as solvent in the presence of 3g HOBt and 5mL DIC; after the reaction was completed, the resin was washed and the solvent was removed.
The N-terminal Fmoc group of the peptidyl resin was deprotected and Fmoc was removed twice with 20% pipeidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
10g of Palm-OH was coupled to the peptidyl Resin using DMF as solvent in the presence of 7g HOBt and 9mL DIC, the reaction was continued for 2.5h, the Resin was washed, the solvent was removed, and after shrink drying 23g of Palm-Lys (Boc) -Val-Lys (Boc) -Leu-Tyr (tBu) -Wang Resin was obtained.
2.4 Cleavage of
Measuring 131mL of TFA, 3.5mL of TIS and 3.5mL of water, uniformly mixing and stirring to obtain a lysate, sealing and placing in a refrigerator at-18 ℃ for later use; the isopropyl ether is placed in a refrigerator at the temperature of minus 18 ℃ for refrigeration for standby.
23g of Palm-Lys (Boc) -Val-Lys (Boc) -Leu-Tyr (tBu) -Wang Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtration, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times, and vacuum drying to obtain 13g Palm-Lys-Val-Lys-Leu-Tyr-OH crude peptide.
2.5 Purification
13g of the crude peptide was weighed and dissolved in 220mL of a mixed solution of acetic acid, methanol and water, and filtered with a microporous membrane having a pore size of 0.22 μm to give a clear and transparent solution, which was purified by reverse phase HPLC, the purification gradient being as shown in the following Table:
time (min) Flow rate (mL/min) A% (acetonitrile) B% (0.1% acetic acid + pure water)
0 40 25 75
15 40 35 65
30 40 45 55
45 40 60 40
80 40 65 35
100 40 65 35
And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (4) Palm-Lys-Val-Lys-Leu-Tyr-OH with the purity of 97.615%.
Example 3
Other peptides of formula (I) of the present invention can be prepared by methods similar to those of example 1 and example 2. The peptides obtained were determined for their molecular weight by ESI-MS.
The results of the peptide (3) test are shown in FIG. 1, which shows, [ M+H ]] + The mass to charge ratio (m/z) of the excimer ion peak was 887.6615 and the mass spectrum measured molecular weight was 886.66.
The results of the peptide (4) test are shown in FIG. 2, which shows, [ M+H ]] + The mass to charge ratio (m/z) of the excimer ion peak was 888.98 and the mass spectrum measured molecular weight was 887.98.
EXAMPLE 4 collagen III content test
4.1 Reagents and materials
Fetal bovine serum, DMEM medium, PBS, DMSO, trypsin, RIPA lysate, collagen III ELISA kit, BCA protein kit.
4.2 Instrument for measuring and controlling the intensity of light
Enzyme-labeled instrument, CO 2 Incubator, superclean bench.
4.3 Cell strain
Human Skin Fibroblasts (HSF).
4.4 Sample to be measured
Polypeptide group: peptides (3) and (4) were dissolved in DMSO at a test concentration of 10ppm.
Blank control group: 0.5% DMSO;
UV group: UV radiation, DMSO was added.
4.5 Experimental method
Taking HSF cells in an exponential growth phase and good state, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, and counting 1-4 multiplied by 10 5 Cell suspensions were prepared at each mL.
Cell suspensions were inoculated onto 6-well plates until cells grew to about 80% and a UV photoaging model was established. The blank control group was added with 50 μl DMSO, supplemented with medium to 2000 μl, and no UV irradiation was performed; adding appropriate amount of PBS, repeatedly washing to colorless, adding 50 μl DMSO, and standing at 40mJ/cm 2 And irradiating under a UV lamp, wherein the distance between the lamp source and the culture flask is 15cm. After irradiation, DMSO was discarded, the UV set was added to 2000 μl of DMSO solution and medium, and the polypeptide set was added to 2000 μl of medium and test sample. The blank, UV, and polypeptide groups continued at 37deg.C, 5% CO 2 Incubate in incubator for 48h.
After the culture, the cells were scraped off by using the cells, the mixture was blown off and homogenized, the supernatant was discarded by centrifugation, RIPA lysate was added, homogenized 3 times (30 s/time, 3min apart) by vortexing, centrifuged at 12000rpm for 10min, and the supernatant was aspirated and was subjected to the procedure according to the collagen III ELISA protocol. OD values of each well were measured with a microplate reader at 450nm over 15 min.
4.6 Experimental results
The III type collagen belongs to collagen forming fibers, accounts for 10% -15% of skin, is mainly present at the joint of the dermis layer and the epidermis layer, provides elasticity and stress resistance for the skin, and can stimulate the synthesis and secretion of the I type collagen and promote tissue regeneration or healing, so that the improvement of the III type collagen content has important significance for tightening the skin and recovering the skin function. The experiment adopts a test sample to treat cells after ultraviolet radiation, and detects the content of type III collagen in the corresponding cells so as to determine whether the peptide of the invention can promote the generation of type III collagen.
The effect of the test sample on the collagen III content is shown in fig. 3. The results show that the collagen III content of the UV group is significantly reduced compared to the blank control group; compared with the UV group, the polypeptide groups can obviously improve the content of the collagen III and promote the generation of the type III collagen.
From the results, the peptide of the invention can increase the content of collagen in cells and promote the generation of collagen, thereby increasing skin elasticity and improving skin firmness; can also restore the function of skin or mucous membrane, promote the regeneration or healing of the skin or mucous membrane, and can be widely applied to products for caring the skin or mucous membrane.
Example 5 collagen I content test
5.1 Reagents and materials
Fetal bovine serum, DMEM medium, PBS, DMSO, trypsin, RIPA lysate, collagen I ELISA kit, BCA protein kit.
5.2 instruments
Enzyme-labeled instrument, CO 2 Incubator, superclean bench.
5.3 cell lines
Human Skin Fibroblasts (HSF).
5.4 sample to be tested
Polypeptide group: peptide (3) and peptide (4), dissolved in DMSO, at a test concentration of 10ppm;
blank control group: 0.5% DMSO;
UV group: UV radiation, DMSO was added.
5.5 Experimental method
Taking HSF fibroblasts in an exponential growth phase, adding 0.25% trypsin digestion solution, digesting to enable adherent cells to fall off, and counting 1-4×10 5 Cell suspensions were prepared at each mL.
Inoculating cell suspension into 6-well plateAnd (3) establishing a UV photoaging model until the cells grow to about 80%. The blank control group was added with 50 μl DMSO, supplemented with medium to 2000 μl, and no UV irradiation was performed; adding appropriate amount of PBS, repeatedly washing to colorless, adding 50 μl DMSO, and standing at 40mJ/cm 2 And irradiating under a UV lamp, wherein the distance between the lamp source and the culture flask is 15cm. After irradiation, DMSO was discarded, the UV set was added to 2000 μl of DMSO solution and medium, and the polypeptide set was added to 2000 μl of medium and test sample. The blank, UV, and polypeptide groups continued at 37deg.C, 5% CO 2 Incubate in incubator for 48h.
After the culture, the cells were scraped off by using the cells, the mixture was blown and homogenized, the supernatant was discarded by centrifugation, RIPA lysate was added, homogenized 3 times (30 s/time, 3min apart) by vortexing, centrifuged at 12000rpm for 10min, and the supernatant was aspirated and was subjected to the procedure according to the collagen I ELISA protocol. OD values of each well were measured with a microplate reader at 450nm over 15 min.
5.6 Experimental results
The type I collagen is the most abundant collagen in a human body, accounts for 80% -85% of skin, is in a thick and tightly arranged bundle-shaped structure, has strong tensile resistance, provides stronger supporting structure and supporting force for the skin, and endows the skin with elasticity and toughness, so that the improvement of the content of the type I collagen has important significance for preventing aging and increasing the skin elasticity and compactness. The experiment adopts a test sample to treat cells after ultraviolet radiation, and detects the content of type I collagen in the corresponding cells so as to determine whether the peptide of the invention can promote the generation of the type I collagen.
The effect of the test sample on the collagen I content is shown in fig. 4. The results show that compared with the blank control group, the collagen I content of the UV group is obviously reduced; compared with the UV group, the polypeptide groups can obviously improve the content of the collagen I and promote the generation of the type I collagen.
From this, the peptide of the present invention can increase the collagen content in cells and promote collagen production, thereby increasing skin elasticity, improving skin firmness and delaying skin aging.
Example 6 cell adhesion experiment
6.1 Reagents and materials
Fetal bovine serum, DMEM medium, PBS, penicillin, streptomycin, MTT.
6.2 Instrument for measuring and controlling the intensity of light
Enzyme-labeled instrument, CO 2 Incubator, superclean bench.
6.3 Cell strain
Human keratinocytes (HaCaT).
6.4 sample to be tested
Polypeptide group: peptide (3) and peptide (4) at test concentrations of 10ppm, 50ppm, 100ppm;
blank control group: PBS.
6.5 Experimental method
Culturing frozen HaCaT human keratinocytes according to a ratio of 1: and (5) carrying out passage 2 to about 5 generations, and selecting cells with better growth vigor as experimental objects.
Samples to be tested were added to 96-well plates at 20 μl/well and oven dried at 37 ℃ overnight. After the HaCaT cells with better growth vigor are digested on the next day, the plates are plated with the HaCaT cells with the density of 1 ten thousand per well, and the culture medium is supplemented to 200 mu L, and the plates are plated with 5% CO at 37 DEG C 2 Incubate in incubator for 3h. After the culture is finished, the culture plate is taken out and the culture medium is continuously supplemented until the liquid level just overflows, and the culture plate is sealed by a sealing film to ensure that no bubbles exist. The mixture was turned clockwise for 20min. The original medium was discarded, 90. Mu.L of fresh medium and 10. Mu.L of 5mg/mL MTT were added to each well, and the mixture was placed at 37℃in 5% CO 2 Incubate in incubator for 3h. The solution was discarded and 150. Mu.L of DMSO was added. Reference OD values at 490nm and 630nm wavelengths were read using a microplate reader.
6.6 Experimental results
Cell adhesion experiments reflect the elasticity of cells by evaluating their ability to adhere. After three-dimensional force action, cells with strong adhesiveness can be kept on a 96-well plate, and the adhesion effect of the cells can be reflected by carrying out MTT quantitative analysis on living cells on the plate. The more viable cells that remain on the 96-well plate, the greater the OD value measured, indicating that the more potent the test sample in promoting cell adhesion, the greater the effect of increasing cell elasticity.
The effect of the test sample on the adhesion of HaCaT cells is shown in FIG. 5, and the result shows that the peptide of the invention can improve the adhesion capacity of keratinocytes and is beneficial to improving the cell elasticity.
Therefore, the peptide can improve the cell adhesion capability, remarkably increase the cell adhesion and the cell-extracellular matrix adhesion, thereby increasing the skin elasticity, improving the skin firmness, delaying the skin aging and improving the skin relaxation problem.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all such alterations and modifications as fall within the scope of the embodiments of the invention.
Finally, it is further noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article or terminal device comprising the element.
The above description has been made in detail for the novel use of the pentapeptides provided by the present invention, and specific examples are employed herein to illustrate the principles and embodiments of the present invention, the above examples being only for aiding in understanding the methods of the present invention and the core ideas thereof; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present invention, the present description should not be construed as limiting the present invention in view of the above.

Claims (9)

1. Use of a peptide of formula (I) or a salt thereof for the preparation of a composition for caring skin or mucous membrane, including one or more of restoring function of skin or mucous membrane, promoting regeneration or healing of skin or mucous membrane, promoting collagen production, increasing skin elasticity or improving skin firmness,
R 1 -Lys-Val-Lys-Leu-Tyr-R 2 （I）
in the formula (I) of the present invention,
R 1 is palmitoyl; r is R 2 is-OH or-NH 2 。
2. The use according to claim 1, wherein the composition comprises a peptide of formula (I) or a salt thereof in a concentration of 0.0001% to 5% by mass;
or, the composition comprises a peptide shown in a formula (I) or a salt thereof with a mass percentage concentration of 0.0005% -1%;
or, the composition comprises the peptide shown in the formula (I) or the salt thereof with the mass percentage concentration of 0.001% -0.1%;
or the composition comprises the peptide shown in the formula (I) or the salt thereof with the mass percentage concentration of 0.005-0.01%.
3. The use according to claim 1, wherein the salt of the peptide of formula (I) comprises a metal salt of the peptide of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.
4. The use according to claim 1, wherein the salt of the peptide of formula (I) comprises a salt of the peptide of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.
5. The use according to claim 1, wherein the salt of the peptide of formula (I) comprises a salt of the peptide of formula (I) with an inorganic or organic acid comprising: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.
6. The use according to claim 1, wherein the peptide of formula (I) or a salt thereof is incorporated into a delivery system or a slow release system or adsorbed onto a solid organic polymer or a solid inorganic support;
the delivery system or sustained release system comprises: liposomes, oleosomes, ethosomes, millimeter capsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, clathrates, lipid vesicles, micelles, millimeter spheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, millimeter particles, microparticles or nanoparticles;
the solid organic polymer or solid inorganic support comprises: talc, bentonite, silica, starch or maltodextrin.
7. The use according to claim 1, wherein the formulation of the composition comprises: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, solution, suspension, emulsion, elixir, polysaccharide film, or jelly.
8. The use according to claim 1, wherein the composition further comprises at least one active substance.
9. The use according to claim 8, wherein the active substance comprises peptides, natural plant ingredients, vitamin C derivatives or retinoids.
CN202311667359.XA 2023-12-07 2023-12-07 Novel use of pentapeptides Pending CN117357426A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116284241B (en) * 2023-04-20 2023-10-31 深圳市维琪科技股份有限公司 Peptides, compositions and uses thereof
CN117327150A (en) * 2023-04-20 2024-01-02 深圳市维琪科技股份有限公司 Peptides, compositions and uses thereof

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