CN116731106A - Synthetic hexapeptide, composition and application thereof - Google Patents

Synthetic hexapeptide, composition and application thereof Download PDF

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Publication number
CN116731106A
CN116731106A CN202310569001.7A CN202310569001A CN116731106A CN 116731106 A CN116731106 A CN 116731106A CN 202310569001 A CN202310569001 A CN 202310569001A CN 116731106 A CN116731106 A CN 116731106A
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ile
agents
trp
asp
peptide
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丁文锋
观富宜
肖玉
赵文豪
孙新林
陈雪
彭晏
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Shenzhen Weiqi Technology Co ltd
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Shenzhen Weiqi Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin

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  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Birds (AREA)
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  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides a compound of formula (I) R 1 ‑X 1 ‑X 2 ‑Asp‑X 3 ‑Ile‑Trp‑R 2 Or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition thereof, and use thereof in the preparation of a whitening composition.

Description

Synthetic hexapeptide, composition and application thereof
Technical Field
The invention relates to the technical field of polypeptides, in particular to a synthetic hexapeptide, a composition containing the peptides and application thereof.
Background
Endothelin (ET) is a biologically active polypeptide initially isolated and purified from cultured porcine aortic endothelial cells, which consists of 21 AAs, has extremely potent vasoconstrictor activity and promotes cell mitosis and cytoblast-like properties, and is associated with a number of physiological pathological conditions such as hypertension, myocardial dysfunction, heart failure, renal insufficiency, renal failure, diabetes, cerebrovascular disease, etc. Under the stimulation of endogenous hormones, vasoactive factors or mechanical stress, endothelial cells synthesize preproendothelin, which is then hydrolyzed by endopeptidase to produce preproendothelin (large endothelin), which is then produced under the action of endothelin converting enzyme. ET mainly comprises three isomers of ET-1, ET-2 and ET-3, wherein ET-1 is mainly expressed in vascular endothelial cells and smooth muscle cells, tracheal epithelial cells, macrophages, fibroblasts, cardiac muscle cells, cerebral neurons and islets; ET-2 is expressed predominantly in ovarian and small intestine epithelial cells; ET-3 is predominantly expressed in endothelial cells and small intestine epithelial cells, while ET-3 also mediates release of vasodilators NO and prostacyclin. The ET-1 effect is the most prominent and is paid attention to.
Endothelin plays a role in physiological pathological processes by binding to endothelin receptors (ETRs). Three classes of endothelin receptors, ETRA, ETRB and ETRC, which are members of the G-protein coupled receptor family, have been discovered to date, and are widely distributed in various tissues and cells in humans, but at varying levels of expression. ETRA is mainly found in heart, aorta and cerebrovascular smooth muscle cells (VSMC), exerting vasoconstriction, promotion of cell proliferation and tissue fibrosis etc.; ETRB is present in endothelial and vascular smooth muscle cells; ETRC is then predominantly distributed in endothelial cells. At present, a plurality of pigment diseases and cardiovascular and cerebrovascular diseases are clinically closely related to the expression of endothelin, for example, the content of the endothelin is increased in pigment accumulation diseases such as freckle-like nevi and chloasma of middle-aged and elderly people, and the endothelin is a substance for strongly constricting blood vessels, and long-time high endothelin level can cause vascular constriction to cause cardiovascular and cerebrovascular diseases; the inhibition of endothelin action is important for the treatment of the above-mentioned diseases, because up-regulation of endothelin expression occurs in most malignant melanoma cells in skin tissues affected by systemic sclerosis.
From the point of view of the synthesis of endothelin, inhibiting the action of endothelin for clinical therapeutic purposes can be considered from the following aspects: (1) inhibiting mRNA expression of pro-endothelin; (2) inhibiting endopeptidase activity such that preproendothelin cannot be hydrolyzed to large endothelin; (3) inhibiting endothelin converting enzyme activity, so that large endothelin cannot be converted into endothelin; (4) antagonizing endothelin receptor and directly inhibiting the action terminal of endothelin. At present, inhibition of endothelin action is typically achieved using endothelin receptor antagonists. Endothelin receptor antagonists are also classified as peptide and non-peptide active substances. The earliest peptide antagonists include BQ123 and TAK-004, but the peptide drugs have limited development due to short half-life in vivo, low oral efficiency, low yield and the like. Then, the non-peptide endothelin receptor antagonists are divided into sulfonamide pyrimidine, amino naphthalene sulfonamide and indane derivatives according to the structure, and the non-peptide ET receptor antagonists have long half-life and are orally effective and are clinically used nowadays. However, there is still a lack of research on high-safety small molecule active polypeptide antagonists. Therefore, there remains a need for the development of a stable, safe, and highly potent polypeptide endothelin receptor antagonist against the mechanism of action of endothelin, thereby inhibiting the action of endothelin.
In skin tissues, endothelin acts as a bridge for interaction between keratinocytes and melanocytes, and is involved in processes such as melanocyte development and melanin synthesis. The growth process of melanocytes is generally undergone 3 stages of neural crest cells, melanocytes and melanocytes, and endothelin is an effective promoter of neural crest cell differentiation to mature melanocytes. In recent years, it has been found that cultured human keratinocytes can synthesize and secrete ET-1 at different concentrations and bind to ET-1 receptor with high affinity on melanocyte membrane, thereby promoting proliferation of melanocyte and enhancing tyrosinase activity, and increasing synthesis of melanin. The increased amount of ET-1 released by keratinocytes in the skin under UV irradiation, which upon acceptance by melanocyte receptors stimulates melanocyte proliferation and tyrosinase activity, thus increasing melanin synthesis. These studies indicate that ET-1 can bind to a specific receptor on the melanocyte membrane, stimulate proliferation and differentiation of melanocytes, activate tyrosinase activity, and thus greatly increase melanin synthesis. It is thus found that endothelin not only has a strong vasoconstrictor effect, but also has an effect on melanocyte proliferation and signal transduction.
The content and distribution of melanin is a major factor in determining skin color. When excessive melanin is accumulated in the human body, the phenomenon of darkening and darkening of the skin is caused, and even uneven melanin distribution on the surface of the skin and plaque are caused. With the development of economy and society and the improvement of living standard, people pay more attention to the maintenance of skin, and for thousands of years women want skin to be tender and white, so that melanin pigmentation and color spot formation become hot spots of modern medical cosmetology, and the development of whitening and freckle removing products and functional raw materials thereof has been in a situation of becoming active and growing rapidly. At present, various whitening products have been developed in order to keep the skin white, prevent the skin from darkening and form color spots. Such as scrub, fruit acid, hydrogen peroxide, arbutin, kojic acid, L-ascorbic acid, nicotinamide, green tea extract or glycoprotein inhibitors, etc. However, these products often have greater cytotoxicity, skin irritation, instability, etc., or are poorly single-acting. Therefore, aiming at the melanin generation mechanism, the development of a novel green, safe, efficient and stable whitening active raw material is particularly necessary.
Disclosure of Invention
In order to overcome the defects in the prior art, the inventor of the invention researches a stable, safe and efficient polypeptide endothelin receptor antagonist by taking an endothelin receptor as an action target point. The peptides and the composition containing the peptides can be combined with endothelin receptor in a competitive way, so that proliferation promoting and regulation effects of endothelin on melanocytes are inhibited, whitening and freckle removing effects are realized, and the peptides and the composition containing the peptides can be widely applied to whitening and freckle removing products and can also be used for preparing opacifiers.
In view of this, the present invention provides a peptide represented by the formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof,
R 1 -X 1 -X 2 -Asp-X 3 -Ile-Trp-R 2 (I)
in the formula (I) of the present invention,
X 1 selected from: -D-His-, -D-Phe-, -D-Trp-, -His-, -Phe-or-Trp-;
X 2 selected from: -Glu-, -Asp-, -Arg-, -Lys-or-His-;
X 3 selected from: -Ile-, -Ala-, -Gly-, -Leu-, -Pro-, -Val-or-Met-;
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 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 [ ]Such as 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, myristoyl, 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, 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 acetyl; r is R 2 is-OH or-NH 2 。
Alternatively, the peptide represented by formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, is selected from the following peptides (1) to (45):
(1)H-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(2)Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(3)H-D-His-Glu-Asp-Ile-Ile-Trp-NH 2 ；
(4)Ac-D-His-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(5)H-D-His-Glu-Asp-Gly-Ile-Trp-OH；
(6)Ac-D-His-Glu-Asp-Leu-Ile-Trp-OH；
(7)Ac-D-His-Glu-Asp-Leu-Ile-Trp-NH 2 ；
(8)H-D-His-Asp-Asp-Ile-Ile-Trp-OH；
(9)H-D-His-Asp-Asp-Ala-Ile-Trp-NH 2 ；
(10)Ac-D-His-Asp-Asp-Val-Ile-Trp-NH 2 ；
(11)Ac-D-His-Asp-Asp-Val-Ile-Trp--OH；
(12)H-D-His-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(13)Ac-D-His-Arg-Asp-Leu-Ile-Trp-OH；
(14)H-D-His-Arg-Asp-Val-Ile-Trp-OH；
(15)Ac-D-His-Arg-Asp-Leu-Ile-Trp-NH 2 ；
(16)H-D-His-Arg-Asp-Val-Ile-Trp-NH 2 ；
(17)H-D-His-Lys-Asp-Leu-Ile-Trp-OH；
(18)H-D-His-Lys-Asp-Gly-Ile-Trp-NH 2 ；
(19)Ac-D-Phe-Glu-Asp-Ile-Ile-Trp-OH；
(20)Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH；
(21)H-D-Phe-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(22)H-D-Phe-Glu-Asp-Val-Ile-Trp-OH；
(23)Ac-D-Phe-Glu-Asp-Leu-Ile-Trp-NH 2 ；
(24)Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(25)H-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(26)Ac-D-Phe-Asp-Asp-Ala-Ile-Trp-NH 2 ；
(27)H-D-Phe-Asp-Asp-Met-Ile-Trp-OH；
(28)H-D-Phe-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(29)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH；
(30)H-D-Phe-Arg-Asp-Ala-Ile-Trp-OH；
(31)Ac-D-Phe-His-Asp-Leu-Ile-Trp-OH；
(32)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(33)H-D-Phe-His-Asp-Leu-Ile-Trp-NH 2 ；
(34)H-D-Trp-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(35)Ac-D-Trp-Glu-Asp-Leu-Ile-Trp-OH；
(36)Ac-D-Trp-Asp-Asp-Ile-Ile-Trp-OH；
(37)H-D-Trp-Asp-Asp-Leu-Ile-Trp-OH；
(38)H-D-Trp-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(39)Ac-D-Trp-Arg-Asp-Gly-Ile-Trp-OH；
(40)H-D-Trp-Arg-Asp-Leu-Ile-Trp-OH；
(41)H-D-Trp-Lys-Asp-Ile-Ile-Trp-OH；
(42)H-D-Trp-Lys-Asp-Met-Ile-Trp-NH 2 ；
(43)Ac-D-Trp-His-Asp-Ile-Ile-Trp-OH；
(44)Ac-D-Trp-HisAsp-Ala-Ile-Trp-NH 2 ；
(45)H-D-Trp-His-Asp-Val-Ile-Trp-OH。
alternatively, the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, is selected from the group consisting of peptide (2), peptide (20), peptide (24), and peptide (29); in particular, the method comprises the steps of,
(2)Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(20)Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH；
(24)Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(29)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-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.
For example, when-Glu-is described herein, it is understood that-Glu-is selected from-L-Glu-, -D-Glu-, or a mixture of both, is racemic or non-racemic. The preparation methods described in this document enable one of ordinary skill in the art to obtain each stereoisomer of the peptides of the invention by selecting amino acids with the correct configuration.
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 at least one atom is replaced by another atom of the same atomic number within the peptide of the invention, but the atomic mass of said other atom is different from the atomic mass normally or predominantly present in nature. Examples of isotopes that can be incorporated into the peptides of the invention are: those of hydrogen, carbon, nitrogen, oxygen or sulfur, e.g. 2 H (deuterium), 3 H (tritium), 13 C、 14 C、 15 N、 17 O、 18 O、 33 S、 34 S、 35 S or 36 S, S. 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.
The term "salt" refers to a salt that is 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.; including salts of peptides of formula (I) with organic bases including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine and the like; including salts of peptides of formula (I) with inorganic or organic acids 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 acid includes: hydrochloric acid, sulfuric acid, boric acid or carbonic acid.
The synthesis of the peptide of formula (I) or a stereoisomer or a salt thereof according to the present invention can be carried out according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis or a combination of solid and liquid phases, and can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of 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.
The functional groups of the side chains of these amino acids remain fully protected with temporary or permanent protecting groups throughout the synthesis and may be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric 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.
In another aspect of the present invention, there is provided a composition comprising an effective amount of a peptide of formula (I) as described above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, and at least one excipient and optionally an adjuvant;
optionally, the adjuvant is selected from: collagen synthesis stimulators, agents that modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulating or inhibiting agent, whitening or depigmenting agent, pigmentation promoting agent, self-tanning agent, antiaging agent, NO-synthase inhibitor, 5α -reductase inhibitor, and pigment inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or agents against atmospheric pollution, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gel polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye pocket, keratolytic agents, antimicrobial agents, antifungal agents, sterilizing agents, bacteriostats, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin synthesis, agents that stimulate cAMP synthesis, agents that stimulate HSP70 synthesis, agents that stimulate heat shock protein synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate deacetylase synthesis, agents that stimulate synthesis of lipid and stratum corneum components, ceramides, fatty acids, agents that inhibit collagen degradation, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, acne dissolvants, anti-psoriasis agents, anti-eczema agents, DNA repair agents, DNA protectants stabilizers, antipruritics, agents for treating and/or caring for sensitive skin, solidifying agents, tightening agents, restructuring agents, stretch-proofing agents, agents for regulating sebum production, antiperspirant agents, agents for stimulating healing, agents for assisting healing, agents for stimulating re-epithelialization, agents for assisting re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents for acting on capillary circulation and/or microcirculation, agents for stimulating angiogenesis, agents for inhibiting vascular permeability, venous tension agents, agents for acting on cellular metabolism, agents for improving dermal-epidermal junction, agents for inducing hair growth, hair growth inhibition or retardation agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents from biofermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against A and/or B ultraviolet light, or mixtures thereof.
Optionally, the formulation of the composition is selected from: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly, or gelatin.
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.
The effective amount of the peptides of the invention to be administered, as well as their dosage, will depend on a number of factors, including the age, the state of the user, the severity of the condition, the route and frequency of administration, and the particular nature of the peptide to be used.
By "effective amount" is meant an amount of one or more peptides of the invention that is nontoxic but sufficient to provide the desired effect. The peptides of the invention are used in the compositions of the invention in concentrations effective to achieve the desired effect; in a preferred form, between 0.00000001% and 20% by weight, preferably between 0.000001% and 15% by weight, more preferably between 0.0001% and 10% by weight, and even more preferably between 0.0001% and 5% by weight, relative to the total weight of the composition.
In another aspect of the present invention, there is provided a delivery system or a sustained release system for achieving better permeation of an active ingredient, which comprises an effective amount of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers, or a salt thereof, or a composition thereof.
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 slow release systems are liposomes, oleosomes, nonionic surfactant liposome 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.
In another aspect of the present invention, there is provided the use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition thereof, or a delivery system or a sustained release system as described above, for the preparation of an endothelin receptor antagonist.
In another aspect, the present invention provides a use of the peptide represented by the above formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or the above composition, or the above delivery system or sustained release system, in the preparation of a whitening composition.
The whitening includes inhibiting tyrosinase activity, inhibiting melanogenesis, lightening skin color, removing color stains, and/or eliminating skin tone non-uniformity.
In another aspect, the present invention provides a use of a peptide of formula (I) above, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition thereof, or a delivery system or a sustained release system as described above, in the preparation of an opacifying agent.
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 ofBiochemical Nomenclature).
Thus, for example, val represents NH 2 -CH(CH(CH 3 ) 2 ) -COOH, val-represents NH 2 -CH(CH(CH 3 ) 2 ) -CO-, -Val represents-NH-CH (CH) 3 ) 2 ) -COOH, and-Val-represents-NH-CH (CH) 3 ) 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 "Ac-" is used in the present invention to denote acetyl (CH) 3 -CO-)。
The beneficial effects obtained by the invention relative to the prior art include:
1. the peptide is obtained through manual design, is convenient to synthesize, and is safe and non-irritating to human bodies.
2. The peptide can be competitively combined with an endothelin receptor, so that the endothelin is inhibited to play a role, the damage of the endothelin to various tissues and organs is reduced, and the peptide can be used for preparing an endothelin receptor antagonist.
3. The peptide provided by the invention can be competitively combined with an endothelin receptor, can inhibit proliferation-promoting regulation effect of the endothelin on melanocytes and inhibit activity of tyrosinase, so that melanin generation is inhibited, skin color is brightened, spots are faded or uneven skin color is eliminated, whitening and spot-removing effects are realized, and the peptide can be widely applied to whitening and spot-removing products and can also be used for preparing opacifiers.
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 schematic diagram of the peptide (2) Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH (formula C) 40 H 59 N 9 O 12 ) Mass spectrum, [ M+H ]] + The mass-to-charge ratio (m/z) of the excimer ion peak was 854.4331, and the mass spectrum measured molecular weight was 853.43;
FIG. 2 is a peptide (20) Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH (formula C) 40 H 51 N 7 O 12 ) Mass spectrum, [ M+Na ]] + The mass-to-charge ratio (m/z) of the summed ion peak was 844.3693 and the mass spectrum measured molecular weight was 821.37;
FIG. 3 shows the peptide (24) Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH (formula C) 42 H 55 N 7 O 12 ) Mass spectrum, [ M+Na ]] + The mass to charge ratio (m/z) of the summed ion peak was 872.4115 and the mass spectrum measured was 849.41.
FIG. 4 is a peptide (29) Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH (formula C) 42 H 55 N 7 O 12 ) Mass spectrum, [ M+H ]] + The mass to charge ratio (m/z) of the excimer ion peak was 891.4980 and the mass spectrum measured molecular weight was 890.50.
FIG. 5 is a graph of the effect of test samples on B16F10 cell melanogenesis.
FIG. 6 is a graph showing the effect of a test sample on tyrosinase activity.
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 any inventive effort, are intended to be within the scope of the invention.
In the present invention, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Biochemical nomenclature Commission in Eur.J.biochem. (1984) 138:9-37 and J.chem. (1989) 264:633-673.
Wang Resin: a starting resin for polypeptide synthesis; DMF: n, N-dimethylformamide; HOBt: 1-hydroxybenzotriazole; DIC: diisopropylcarbodiimide; DIPEA: diisopropylethylamine; ac (Ac) 2 O: acetic anhydride; piperidine: piperidine; TFA: trifluoroacetic acid; TIS: triisopropylsilane; EDT:1, 2-ethanedithiol; asp: aspartic acid; glu: glutamic acid; his: histidine; lie: isoleucine; trp: tryptophan; phe: phenylalanine; ala: alanine; arg: arginine; fmoc: 9-fluorenylmethoxycarbonyl; boc: a tert-butoxycarbonyl group; otBu: t-butoxy; trt: a trityl group; pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl.
Example 1 computer simulation
Endothelin receptor antagonists inhibit the proliferation-promoting regulatory effect of endothelin on melanocytes primarily through competitive binding to endothelin receptors. Based on this, candidate peptides were screened by molecular docking of polypeptides to endothelin receptors using in silico methods and analysis of their binding patterns to the receptors. The results of molecular docking scoring of polypeptides with endothelin receptors are shown in Table 2 below.
Table 2 molecular docking scoring of polypeptides and endothelin receptors
Sequence(s) Score value Binding energy (kcal/mol)
Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH 11.70 536.57
Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH 12.23 526.86
Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH 14.43 537.76
Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH 10.59 543.96
The results show that the peptide of the invention can be well combined with an endothelin receptor, thereby antagonizing the endothelin receptor and inhibiting the proliferation promoting and regulating effects of the endothelin on melanocytes, and can be used for preparing endothelin receptor antagonists.
Example 2 preparation of Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH
2.1 swelling of resin
6g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the resin and the solvent was removed.
2.2 feeding reaction
8.1g of Fmoc-Trp (Boc) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. 6.3mL DIC was added to activate for 3min to avoid water vapor. Adding the activated amino acid into the swelled resin for reaction for 2 hours, pumping away the reaction solution, and continuously adding Ac 2 O and DIPEA are blocked for 3 hours. The resin was washed and the solvent was pumped away. K detection resin is colorless and transparent, which indicates that the reaction is complete.
Deprotection of the N-terminal Fmoc group was performed and 3.8g of activated Fmoc-Ile-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 4.4g HOBt and 6.3mLDIC, and the reaction was continued 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, 3.8g of Fmoc-Ile-OH, 5.4g of Fmoc-Asp (OtBu) -OH, 5.5g of Fmoc-Glu (OtBu) -OH and then 9.3g of Fmoc-D-His (Trt) -OH were coupled sequentially using DMF as solvent in the presence of 4.4g of HOBt and 6.3mL 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% piperidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
In the presence of DIPEA, 8.5g of Ac was treated with DMF as solvent 2 O is coupled to the peptide-based resin, the reaction is continued for 1.5h, the resin is washed, the solvent is removed, and 16.0g of Ac-D-His (Trt) -Glu (OtBu) -Asp (OtBu) -Ile-Ile-Trp (Boc) -WangResin is obtained after shrinkage drying.
2.3 cleavage
Weighing 86.4mL of TFA, 2.4mL of TIS, 2.4mL of EDT, 2.4mL of phenyl sulfide and 2.4mL of water, mixing and stirring uniformly 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.
16.0g of Ac-D-His (Trt) -Glu (OtBu) -Asp (OtBu) -Ile-Ile-Trp (Boc) -Wang Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times until the pH value is 3-4, and vacuum drying to obtain 6.8g Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH crude peptide.
2.4 purification
Weigh 6.8g of crude peptide in 300mL methanol: water: in acetic acid (V: v=1:2:1), a clear and transparent solution was obtained by filtration through a microporous membrane with a pore size of 0.45 μm, and purified by reverse phase HPLC with the following purification gradient:
And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (2) Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH with the purity of 98.21%.
EXAMPLE 3 preparation of Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH
3.1 swelling of resin
6g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the resin and the solvent was removed.
3.2 feeding reaction
8.1g of Fmoc-Trp (Boc) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. 6.3mL DIC was added to activate for 3min to avoid water vapor. Adding the activated amino acid into the swelled resin for reaction for 2 hours, pumping away the reaction solution, and continuously adding Ac 2 O and DIPEA are blocked for 3 hours. The resin was washed and the solvent was pumped away. K detection resin is colorless and transparent, which indicates that the reaction is complete.
Deprotection of the N-terminal Fmoc group was performed and 3.8g of activated Fmoc-Ile-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 4.4g HOBt and 6.3mLDIC, and the reaction was continued 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, 3.4g Fmoc-Ala-OH, 5.4g Fmoc-Asp (OtBu) -OH, 5.5g Fmoc-Glu (OtBu) -OH and then 5.8g Fmoc-D-Phe-OH were coupled sequentially using DMF as solvent in the presence of 4.4g HOBt and 6.3 mLDIC; 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% piperidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
In the presence of DIPEA, 8.5g of Ac was treated with DMF as solvent 2 O is coupled to the peptide-based Resin, the reaction is continued for 1.5h, the Resin is washed, the solvent is removed, and after shrinkage drying, 15.0g of Ac-D-Phe-Glu (OtBu) -Asp (OtBu) -Ala-Ile-Trp (Boc) -Wang Resin is obtained.
3.3 cleavage
Weighing 86.4mL of TFA, 2.4mL of TIS, 2.4mL of EDT, 2.4mL of phenyl sulfide and 2.4mL of water, mixing and stirring uniformly 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.
15.0g of Ac-D-Phe-Glu (OtBu) -Asp (OtBu) -Ala-Ile-Trp (Boc) -Wang Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times until the pH value is 3-4, and vacuum drying to obtain 7.3g Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH crude peptide.
3.4 purification
7.3g of crude peptide was weighed out and dissolved in 300mL of methanol: water: in acetic acid (V: v=1:2:1), a clear and transparent solution was obtained by filtration through a microporous membrane with a pore size of 0.45 μm, and purified by reverse phase HPLC with the following purification gradient:
Time (min) Flow rate (mL/min) A% (0.1% acetic acid + acetonitrile) B% (0.1% acetic acid + pure water)
0 40 20 80
15 40 65 35
20 40 75 25
21 40 20 80
And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (20) Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH with the purity of 97.35%.
EXAMPLE 4 preparation of Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH
4.1 swelling of resin
6g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the resin and the solvent was removed.
4.2 feeding reaction
8.1g of Fmoc-Trp (Boc) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. Adding 6.3mL DIC to activate for 3min to avoid water vapor. Adding the activated amino acid into the swelled resin for reaction for 2 hours, pumping away the reaction solution, and continuously adding Ac 2 O and DIPEA are blocked for 3 hours. The resin was washed and the solvent was pumped away. K detection resin is colorless and transparent, which indicates that the reaction is complete.
Deprotection of the N-terminal Fmoc group was performed and 3.8g of activated Fmoc-Ile-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 4.4g HOBt and 6.3mLDIC, and the reaction was continued 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, 3.8g of Fmoc-Ile-OH, 5.4g of Fmoc-Asp (OtBu) -OH and then 5.8g of Fmoc-D-Phe-OH were coupled sequentially using DMF as solvent in the presence of 4.4g of HOBt and 6.3mL 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% piperidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
In the presence of DIPEA, 8.5g of Ac was treated with DMF as solvent 2 O was coupled to the peptidyl Resin and the reaction was continued for 1.5h, the Resin was washed, the solvent was removed, and 13.0g of Ac-D-Phe-Asp (OtBu) -Asp (OtBu) -Ile-Ile-Trp (Boc) -Wang Resin was obtained after shrink drying.
4.3 cleavage
Weighing 86.4mL of TFA, 2.4mL of TIS, 2.4mL of EDT, 2.4mL of phenyl sulfide and 2.4mL of water, mixing and stirring uniformly 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.
13.0g of Ac-D-Phe-Asp (OtBu) -Asp (OtBu) -Ile-Ile-Trp (Boc) -Wang Resin was weighed, added to a round bottom flask, the frozen lysate was added and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times until the pH value is 3-4, and vacuum drying to obtain 5.3g Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH crude peptide.
4.4 purification
Weigh 5.3g of crude peptide in 300mL methanol: water: in aqueous ammonia (V: v=30:30:1), a clear and transparent solution was obtained by filtration through a microporous membrane with a pore size of 0.45 μm, and purified by reverse phase HPLC with the following purification gradient:
Time (min) Flow rate (mL/min) A% (0.1% acetic acid + acetonitrile) B% (0.1% acetic acid + pure water)
0 40 20 80
15 40 65 35
20 40 75 25
21 40 20 80
And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (24) Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH with the purity of 96.6%.
EXAMPLE 5 preparation of Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH
5.1 swelling of resin
6g of Wang resin was weighed into a solid phase synthesis reaction column, swollen with DMF, washed the resin and the solvent was removed.
5.2 feeding reaction
8.1g of Fmoc-Trp (Boc) -OH and 4.4g of HOBt were weighed into a dry flask, dissolved in DMF and sealed in a refrigerator at-18℃for 30min. 6.3mL DIC was added to activate for 3min to avoid water vapor. Adding the activated amino acid into the swelled resin for reaction for 2 hours, pumping away the reaction solution, and continuously adding Ac 2 O and DIPEA are blocked for 3 hours. The resin was washed and the solvent was pumped away. K detection resin is colorless and transparent, which indicates that the reaction is complete.
Deprotection of the N-terminal Fmoc group was performed and 3.8g of activated Fmoc-Ile-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 4.4g HOBt and 6.3mLDIC, and the reaction was continued 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, 3.8g of Fmoc-Ile-OH, 5.4g of Fmoc-Asp (OtBu) -OH, 11.3g of Fmoc-Arg (Pbf) -OH and then 5.8g of Fmoc-D-Phe-OH were coupled sequentially using DMF as solvent in the presence of 4.4g of HOBt and 6.3mL 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% piperidine/DMF for 10min each time, sampling K and developing dark blue. The resin was washed 6 times with DMF and the solvent was removed.
In the presence of DIPEA, 8.5g of Ac was treated with DMF as solvent 2 O is coupled to the peptide-based resin, the reaction is continued for 1.5h, the resin is washed, the solvent is removed, and 16.5g of Ac-D-Phe-Arg (Pbf) -Asp (OtBu) -Ile-Ile-Trp (Boc) -Wangresin is obtained after shrinkage and drying.
5.3 cleavage
Weighing 86.4mL of TFA, 2.4mL of TIS, 2.4mL of EDT, 2.4mL of phenyl sulfide and 2.4mL of water, mixing and stirring uniformly 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.
16.5g of Ac-D-Phe-Arg (Pbf) -Asp (OtBu) -Ile-Ile-Trp (Boc) -Wangresin was weighed, added to a round bottom flask, the frozen lysate was added, and the reaction was stirred for 2h. Suction filtering, collecting filtrate, concentrating to 15mL, adding isopropyl ether, stirring, centrifuging and washing for 6 times until the pH value is 3-4, and vacuum drying to obtain 4.8g Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH crude peptide.
5.4 purification
Weigh 4.8g of crude peptide in 300mL methanol: water: in aqueous ammonia (V: v=30:30:1), a clear and transparent solution was obtained by filtration through a microporous membrane with a pore size of 0.45 μm, and purified by reverse phase HPLC with the following purification gradient:
Time (min) Flow rate (mL/min) A% (0.1% acetic acid + acetonitrile) B% (0.1% acetic acid + pure water)
0 40 20 80
15 40 65 35
20 40 75 25
21 40 20 80
And (3) purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (29) Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH with the purity of 99.1%.
Example 6
Other peptides of formula (I) of the present invention may be prepared by similar methods.
The peptides obtained were determined by ESI-MS for molecular weight and the test results for some compounds are shown in Table 3 below and FIGS. 1-4:
table 3 mass spectrometry determination of molecular weight results
Numbering device Sequence(s) Molecular weight mass spectrometry analysis results
(2) Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH 853.43
(20) Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH 821.37
(24) Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH 849.41
(29) Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH 890.50
Example 7 Effect on melanogenesis in murine melanoma cells (B16F 10)
7.1 reagents and materials
DMEM medium, fetal bovine serum, trypsin, naOH, DMSO.
7.2 instruments
Enzyme-labeled instrument (MD, CO) 2 Incubator (Shanghai-constant), ultra clean bench (su zhou purification).
7.3 cell lines
Mouse melanoma cell line (B16F 10) was purchased from Shanghai cell bank, national academy of sciences.
7.4 sample to be tested
Sample group: peptide (2), peptide (20), peptide (24), peptide (29) and arbutin were tested at concentrations of 12.5ppm, 25ppm, 50ppm;
blank control group: PBS.
7.5 Experimental methods
Reference standard: T/SHRH 027-2019 in vitro test B16 cell melanin synthesis inhibition experiments.
Taking one bottle of cells in good exponential growth phase, adding 0.25% trypsin digestion solution to make the adherent cells fall off, counting 1-4×10 5 Cell suspensions were prepared at each mL.
The medium in the wells was completely aspirated and starved by adding 1ml of LPBS solution for 3-6h.
And setting the concentration of the sample, diluting the concentration of the sample according to the dilution multiple, and adding the sample to be tested.
The culture medium is replaced every 24 to 48 hours,after culturing for 72 hours, cells were collected and the cell density was adjusted to 5X 10 5 The supernatant was discarded after centrifugation (4000 r/min,5 min). 450. Mu.L of 10% DMSO and 1M NaOH solution were added and the mixture was placed in a water bath at 80℃for 30min to completely dissolve the cell pellet. The OD of each well was measured with an enzyme-labeled instrument at a wavelength of 490 nm.
Melanin content (%) =sample group OD 490 Blank OD 490 ×100％
7.6 results
The effect of the test sample on B16F10 cell melanogenesis is shown in FIG. 5. The results show that the sample groups can reduce the melanin content within the range of 12.5-50ppm compared with the blank group, wherein the melanin content can be obviously reduced by the peptide (29) at the low concentration of 12.5ppm, the inhibiting effect of the peptide (29) on melanin generation has better effect compared with arbutin at the same concentration, and the inhibiting effect of the peptide (2), the peptide (20) and the peptide (24) on melanin generation is equivalent to that of the arbutin at the same concentration.
As is known, arbutin has high skin irritation, can not be used for a long time, and is easy to cause side effects such as local redness, itching, facial stinging and the like after being used by partial patients. Furthermore, arbutin has quite strict requirements on the use concentration, and the use at high concentration can lead to skin white spots. In contrast, the small molecule active polypeptide has higher safety, is mild and has no stimulation to skin, and the peptide can exert better whitening effect than arbutin under the condition of meeting high safety.
From this, it is clear that the peptide of the present invention can inhibit melanin formation, reduce melanin content, and has a whitening effect.
Example 8 Effect on tyrosinase activity
8.1 reagents and materials
Mushroom tyrosinase, L-DOPA.
8.2 instruments
Microplate reader (MD), ultra clean bench (su zhou clean).
8.3 sample to be tested
Sample group: peptide (2), peptide (20), peptide (24), peptide (29) and arbutin were tested at concentrations of 12.5ppm, 25ppm and 50ppm.
Blank control group: PBS.
8.4 Experimental methods
Sample concentrations are set, tyrosinase 100 mu L (1U/. Mu.L) is added into sample solutions to be measured with different concentrations, and the sample solutions are placed in a water bath at 37 ℃ for 10min. Adding 0.1% L-DOPA solution, continuously heating in water bath at 37deg.C for 10min, and immediately measuring OD at 475nm wavelength of enzyme labeling instrument 475 Values.
Tyrosinase inhibition rate (%) = (1-sample group OD 475 Blank OD 475 )×100％
8.5 results
The effect of the test sample on tyrosinase activity is shown in fig. 6. The results show that in the range of 12.5-50ppm, the sample groups can improve the tyrosinase inhibition rate and inhibit the tyrosinase activity, so that the melanin generation is reduced, wherein the peptide (29) can obviously inhibit the tyrosinase activity at a low concentration of 12.5ppm, compared with arbutin at the same concentration, the inhibition effect of the peptide (29) on the tyrosinase is stronger, and the inhibition effect of the peptide (20) and the peptide (24) on the tyrosinase is equivalent to that of the arbutin at the same concentration.
Therefore, the peptide disclosed by the invention can inhibit the activity of tyrosinase and reduce the generation of melanin, can be used for brightening skin, fading and removing color spots or eliminating uneven skin color, realizes the effect of whitening and removing spots, can be widely applied to products for whitening and removing spots, and can also be used for preparing opacifiers.
EXAMPLE 9 preparation of a microemulsion composition containing peptide (2)
Weighing the component of phase B according to the dosage of the prescription, and adding the component into a container. Then, phase D was added to phase B and homogenized with continuous stirring. Phase a was then added to the mixture. Finally, phase C is added to obtain the micro emulsion composition containing the peptide (2).
Example 10 preparation of essence containing peptide (20)
Stirring and heating the purified water to 85 ℃, and preserving heat for 30min; pre-dissolving sodium hyaluronate and xanthan gum in butanediol, adding into water, stirring and dissolving completely; stirring and cooling to 35 ℃, adding the rest ingredients, and stirring uniformly.
EXAMPLE 11 preparation of liposomes containing peptide (24)
Dipalmitoyl phosphatidylcholine was weighed and dissolved in chloroform. Evaporating the solvent under vacuum until a thin layer of phospholipid is obtained, and hydrating the layer by treating it with an aqueous peptide solution of the desired concentration at 55℃to obtain a multilamellar liposome. The multi-chamber liposome is subjected to high-pressure homogenization treatment to obtain single-chamber liposome with smaller and uniform size.
Example 12 preparation of toner containing peptide (29)
Dissolving allantoin and glycerol with water, heating to 85deg.C, and maintaining for 30min; dissolving PEG-7 glycerol cocoate and peptide (29) with water; the above solutions are cooled and then mixed, and the mixed solution is obtained after uniform stirring; and sequentially adding propylene glycol, preservative and essence into the mixed solution, adding water and stirring uniformly to obtain the product.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, but is not intended to limit the practice of the invention to such description. It will be apparent to those skilled in the art that several simple deductions or substitutions can be made without departing from the spirit of the invention, and the invention is not limited to the above-mentioned embodiments.

Claims (12)

1. A peptide represented by the formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof,
R 1 -X 1 -X 2 -Asp-X 3 -Ile-Trp-R 2 (I)
in the formula (I) of the present invention,
X 1 selected from: -D-His-, -D-Phe-, -D-Trp-, -His-, -Phe-or-Trp-;
X 2 selected from: -Glu-, -Asp-, -Arg-, -Lys-or-His-;
X 3 selected from: -Ile-, -Ala-, -Gly-, -Leu-, -Pro-, -Val-or-Met-;
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 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.
2. The peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, as claimed in claim 1, wherein R 1 Selected from: H. acetyl, t-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, 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, 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 acetyl; r is R 2 is-OH or-NH 2 。
3. The peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, according to claim 1, wherein the peptide is selected from the group consisting of the following peptides (1) to (45):
(1)H-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(2)Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(3)H-D-His-Glu-Asp-Ile-Ile-Trp-NH 2 ；
(4)Ac-D-His-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(5)H-D-His-Glu-Asp-Gly-Ile-Trp-OH；
(6)Ac-D-His-Glu-Asp-Leu-Ile-Trp-OH；
(7)Ac-D-His-Glu-Asp-Leu-Ile-Trp-NH 2 ；
(8)H-D-His-Asp-Asp-Ile-Ile-Trp-OH；
(9)H-D-His-Asp-Asp-Ala-Ile-Trp-NH 2 ；
(10)Ac-D-His-Asp-Asp-Val-Ile-Trp-NH 2 ；
(11)Ac-D-His-Asp-Asp-Val-Ile-Trp--OH；
(12)H-D-His-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(13)Ac-D-His-Arg-Asp-Leu-Ile-Trp-OH；
(14)H-D-His-Arg-Asp-Val-Ile-Trp-OH；
(15)Ac-D-His-Arg-Asp-Leu-Ile-Trp-NH 2 ；
(16)H-D-His-Arg-Asp-Val-Ile-Trp-NH 2 ；
(17)H-D-His-Lys-Asp-Leu-Ile-Trp-OH；
(18)H-D-His-Lys-Asp-Gly-Ile-Trp-NH 2 ；
(19)Ac-D-Phe-Glu-Asp-Ile-Ile-Trp-OH；
(20)Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH；
(21)H-D-Phe-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(22)H-D-Phe-Glu-Asp-Val-Ile-Trp-OH；
(23)Ac-D-Phe-Glu-Asp-Leu-Ile-Trp-NH 2 ；
(24)Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(25)H-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(26)Ac-D-Phe-Asp-Asp-Ala-Ile-Trp-NH 2 ；
(27)H-D-Phe-Asp-Asp-Met-Ile-Trp-OH；
(28)H-D-Phe-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(29)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH；
(30)H-D-Phe-Arg-Asp-Ala-Ile-Trp-OH；
(31)Ac-D-Phe-His-Asp-Leu-Ile-Trp-OH；
(32)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(33)H-D-Phe-His-Asp-Leu-Ile-Trp-NH 2 ；
(34)H-D-Trp-Glu-Asp-Ala-Ile-Trp-NH 2 ；
(35)Ac-D-Trp-Glu-Asp-Leu-Ile-Trp-OH；
(36)Ac-D-Trp-Asp-Asp-Ile-Ile-Trp-OH；
(37)H-D-Trp-Asp-Asp-Leu-Ile-Trp-OH；
(38)H-D-Trp-Arg-Asp-Ile-Ile-Trp-NH 2 ；
(39)Ac-D-Trp-Arg-Asp-Gly-Ile-Trp-OH；
(40)H-D-Trp-Arg-Asp-Leu-Ile-Trp-OH；
(41)H-D-Trp-Lys-Asp-Ile-Ile-Trp-OH；
(42)H-D-Trp-Lys-Asp-Met-Ile-Trp-NH 2 ；
(43)Ac-D-Trp-His-Asp-Ile-Ile-Trp-OH；
(44)Ac-D-Trp-His-Asp-Ala-Ile-Trp-NH 2 ；
(45)H-D-Trp-His-Asp-Val-Ile-Trp-OH。
4. a peptide of formula (I), or a stereoisomer thereof, or a mixture of its stereoisomers, or a salt thereof, according to claim 3, selected from the group consisting of peptide (2), peptide (20), peptide (24), peptide (29); in particular, the method comprises the steps of,
(2)Ac-D-His-Glu-Asp-Ile-Ile-Trp-OH；
(20)Ac-D-Phe-Glu-Asp-Ala-Ile-Trp-OH；
(24)Ac-D-Phe-Asp-Asp-Ile-Ile-Trp-OH；
(29)Ac-D-Phe-Arg-Asp-Ile-Ile-Trp-OH。
5. the peptide of formula (I) according to any one of claim 1 to 4, wherein the peptide is a stereoisomer thereof or a mixture of stereoisomers thereof or a salt thereof,
the salts include metal salts of peptides of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;
alternatively, the salt includes a salt of a peptide of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;
alternatively, the salt includes a salt of a peptide represented by formula (I) with an inorganic acid or an organic acid, the organic acid including: 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. A composition comprising an effective amount of a peptide of formula (I), or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, as defined in any one of claims 1 to 5, together with at least one excipient and optionally an adjuvant;
optionally, the adjuvant is selected from: collagen synthesis stimulators, agents that modulate PGC-1 alpha synthesis, agents that modulate PPARgamma activity, agents that increase or decrease the triglyceride content of adipocytes, agents that stimulate or delay adipocyte differentiation, lipolytic or lipolysis-stimulating agents, lipolytic agents, adipogenic agents, inhibitors of acetylcholine receptor aggregation, agents that inhibit muscle contraction, anticholinergic agents, elastase inhibitors, matrix metalloproteinase inhibitors, melanin synthesis stimulating or inhibiting agent, whitening or depigmenting agent, pigmentation promoting agent, self-tanning agent, antiaging agent, NO-synthase inhibitor, 5α -reductase inhibitor, and pigment inhibitors of lysyl hydroxylase and/or prolyl hydroxylase, antioxidants, free radical scavengers and/or agents against atmospheric pollution, active carbonyl scavengers, anti-glycation agents, antihistamines, antiviral agents antiparasitic agents, emulsifiers, emollients, organic solvents, liquid propellants, skin conditioning agents, moisture retaining substances, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermohydrolases, vitamins, amino acids, proteins, pigments, dyes, biopolymers, gel polymers, thickeners, surfactants, softeners, adhesives, preservatives, anti-wrinkle agents, agents capable of reducing or treating the lower eye pocket, keratolytic agents, antimicrobial agents, antifungal agents, sterilizing agents, bacteriostats, agents that stimulate the synthesis of dermal or epidermal macromolecules and/or that inhibit or prevent their degradation, agents that stimulate elastin synthesis, agents that stimulate decorin synthesis, agents that stimulate laminin synthesis, agents that stimulate defensin synthesis, agents that stimulate chaperonin synthesis, agents that stimulate cAMP synthesis, agents that stimulate HSP70 synthesis, agents that stimulate heat shock protein synthesis, agents that stimulate hyaluronic acid synthesis, agents that stimulate fibronectin synthesis, agents that stimulate deacetylase synthesis, agents that stimulate synthesis of lipid and stratum corneum components, ceramides, fatty acids, agents that inhibit collagen degradation, agents that inhibit elastin degradation, agents that inhibit serine proteases, agents that stimulate fibroblast proliferation, agents that stimulate keratinocyte proliferation, agents that stimulate adipocyte proliferation, agents that stimulate melanocyte proliferation, agents that stimulate keratinocyte differentiation, agents that inhibit acetylcholinesterase, skin relaxants, agents that stimulate glycosaminoglycan synthesis, anti-hyperkeratosis agents, acne dissolvants, anti-psoriasis agents, anti-eczema agents, DNA repair agents, DNA protectants stabilizers, antipruritics, agents for treating and/or caring for sensitive skin, solidifying agents, tightening agents, restructuring agents, stretch-proofing agents, agents for regulating sebum production, antiperspirant agents, agents for stimulating healing, agents for assisting healing, agents for stimulating re-epithelialization, agents for assisting re-epithelialization, cytokines, sedatives, anti-inflammatory agents, anesthetics, agents for acting on capillary circulation and/or microcirculation, agents for stimulating angiogenesis, agents for inhibiting vascular permeability, venous tension agents, agents for acting on cellular metabolism, agents for improving dermal-epidermal junction, agents for inducing hair growth, hair growth inhibition or retardation agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents from biofermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against A and/or B ultraviolet light, or mixtures thereof.
7. The composition of claim 6, wherein the formulation of the composition is selected from the group consisting of: cream, oil, balm, foam, lotion, gel, wipe, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly, or gelatin.
8. A delivery system or sustained release system comprising an effective amount of a peptide of formula (I) according to any one of claims 1 to 5, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition according to claim 6 or 7;
the delivery system or slow release system is selected from: liposomes, oleosomes, nonionic surfactant liposome 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.
9. Use of a peptide of formula (I) according to any one of claims 1 to 5, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition according to claim 6 or 7, or a delivery system or a sustained release system according to claim 8, for the preparation of an endothelin receptor antagonist.
10. Use of a peptide of formula (I) according to any one of claims 1 to 5, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition according to claim 6 or 7, or a delivery system or a slow release system according to claim 8, for the preparation of a whitening composition.
11. The use according to claim 10, wherein the whitening comprises inhibiting tyrosinase activity, inhibiting melanin production, lightening skin colour, removing colour stains and/or eliminating skin colour non-uniformities.
12. Use of a peptide of formula (I) according to any one of claims 1 to 5, or a stereoisomer thereof, or a mixture of stereoisomers thereof, or a salt thereof, or a composition according to claim 6 or 7, or a delivery system or a slow release system according to claim 8, for the preparation of an opacifying agent.
CN202310569001.7A 2023-05-19 2023-05-19 Synthetic hexapeptide, composition and application thereof Pending CN116731106A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117417408A (en) * 2023-12-14 2024-01-19 深圳市维琪科技股份有限公司 Anti-aging hexapeptide, composition and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117417408A (en) * 2023-12-14 2024-01-19 深圳市维琪科技股份有限公司 Anti-aging hexapeptide, composition and application thereof
CN117417408B (en) * 2023-12-14 2024-02-13 深圳市维琪科技股份有限公司 Anti-aging hexapeptide, composition and application thereof

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