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WO2025190245A1 - Polypeptides et leur utilisation - Google Patents

Polypeptides et leur utilisation

Info

Publication number
WO2025190245A1
WO2025190245A1 PCT/CN2025/081717 CN2025081717W WO2025190245A1 WO 2025190245 A1 WO2025190245 A1 WO 2025190245A1 CN 2025081717 W CN2025081717 W CN 2025081717W WO 2025190245 A1 WO2025190245 A1 WO 2025190245A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
pep
amino acid
melanin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2025/081717
Other languages
English (en)
Chinese (zh)
Inventor
佘超民
刘鹤宁
陈雄伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Imeik Technology Development Co Ltd
Original Assignee
Beijing Imeik Technology Development Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Imeik Technology Development Co Ltd filed Critical Beijing Imeik Technology Development Co Ltd
Publication of WO2025190245A1 publication Critical patent/WO2025190245A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides 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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • 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

Definitions

  • the present invention relates to the technical fields of biomedicine, medical devices, cosmetics and functional polypeptides, and in particular to polypeptides having whitening and freckle-removing effects such as inhibiting tyrosinase activity, reducing melanin secretion and resisting oxidation, and their applications.
  • Melanin protects the skin from ultraviolet radiation and inhibits photocarcinogenesis, but excessive melanin production can cause pigmentation, primarily manifesting as freckles, age spots, chloasma, and uneven skin tone.
  • Melanin is a complex, multi-step conversion product of L-tyrosine.
  • Melanin in skin cells is primarily composed of two quinone-type polymers, eumelanin and pheomelanin, and is the primary factor influencing skin color.
  • Tyrosinase is a copper-containing enzyme widely distributed in microorganisms, plants and animals. It regulates plant browning in plants and is responsible for skin and hair pigmentation in animals. It is also a key rate-limiting enzyme in the formation of melanin. Tyrosinase catalyzes the conversion of L-tyrosine into 3,4-dihydroxyphenylalanine (DOPA), and then catalyzes the conversion of DOPA to dopaquinone. Dopaquinone is the main factor affecting the formation of different melanins. Therefore, in theory, inhibiting tyrosinase activity can effectively inhibit the production of melanin.
  • DOPA 3,4-dihydroxyphenylalanine
  • Tyrosinase inhibitors currently on the market include hydroquinone, arbutin, kojic acid, and some electron-rich phenols, which have been shown to effectively inhibit tyrosinase activity.
  • Hydroquinone is unstable and easily oxidized. It was later discovered to be highly irritating to the skin, causing irritation or contact dermatitis. Furthermore, if used inappropriately, it can cause excessive discoloration of the skin, leading to localized whitening. Consequently, hydroquinone has been banned from cosmetics.
  • Arbutin is a derivative of hydroquinone with a similar mechanism of action. Because arbutin is a sugar ligand of hydroquinone, its structural toxicity to melanocytes is significantly reduced.
  • polypeptide sequences exhibit a good inhibitory effect on tyrosinase. This polypeptide competitively binds to the tyrosinase target and exhibits a higher tyrosinase inhibitory effect. At the same time, short peptide sequences can exhibit better transdermal efficacy and higher safety.
  • the present invention provides a group of polypeptides that exhibit strong tyrosinase inhibition, the ability to inhibit melanin production in B16 cells, antioxidant activity, and low cytotoxicity. These polypeptides are suitable for use in the preparation of cosmetics, tissue fillers, pharmaceuticals, foods, or agricultural products. Furthermore, the polypeptides provided by the present invention have a low molecular weight and excellent transdermal properties, making them particularly suitable for use in the cosmetics field.
  • the first aspect of the present invention provides a polypeptide, the amino acid sequence of which is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the second aspect of the present invention provides a tyrosinase inhibitor, which comprises a polypeptide having an amino acid sequence of CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the third aspect of the present invention provides a melanin inhibitor, which comprises a polypeptide, and the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the fourth aspect of the present invention provides an antioxidant, which includes a polypeptide, and the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the fifth aspect of the present invention provides a whitening polypeptide, the amino acid sequence of which is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the sixth aspect of the present invention provides the use of a polypeptide in inhibiting tyrosinase, inhibiting melanin, anti-oxidation, whitening or removing spots, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the seventh aspect of the present invention provides a method for preparing the above-mentioned polypeptide, which is prepared by solid phase synthesis or recombinant expression and purified.
  • the polypeptide is synthesized through artificial solid phase synthesis and purified by desalting on a reverse phase HPLC column or filtering and purifying the crude polypeptide solution using a filter membrane.
  • the purity of the prepared polypeptide is greater than 95%.
  • the preparation method further comprises obtaining the finished powder by freeze-drying technology.
  • the preparation method comprises the following steps:
  • the decapping solution is a DMF solution containing piperidine.
  • the decapping solution is a DMF solution containing piperidine.
  • the cutting solution is a DCM solution containing trifluoroacetic acid TFA.
  • the purified polypeptide product is freeze-dried to obtain a finished product powder.
  • the eighth aspect of the present invention provides the use of a polypeptide in the preparation of cosmetics, foods, agricultural products, medicines, and tissue fillers, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the ninth aspect of the present invention provides a composition comprising a polypeptide and an auxiliary material; wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the auxiliary material is selected from one or more of excipients, diluents, humectants, emulsifiers, pH regulators, thickeners, disintegrants, lubricants, surfactants, suspending agents, gelling agents, preservatives, stabilizers, colorants or fragrances.
  • the composition is a cosmetic, a food, an agricultural product, a medicine or a tissue filler.
  • the composition can treat, prevent or repair skin pigmentation problems, inhibit melanin production, whiten, remove spots, and provide anti-oxidation.
  • the composition is used in preparing products for treating, preventing or repairing skin pigmentation problems, inhibiting melanin production, whitening, removing spots and anti-oxidation.
  • the composition may contain 0.01-99.5% by weight (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the polypeptide.
  • 0.01-99.5% by weight e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%
  • composition can be administered by any suitable route, such as enteral (e.g. oral) or parenteral (e.g. intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.) routes.
  • enteral e.g. oral
  • parenteral e.g. intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.
  • composition can be in any suitable dosage form, including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, injectable preparations, and the like.
  • suitable dosage form including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, injectable preparations, and the like.
  • the tenth aspect of the present invention provides a method for preparing a composition, which comprises mixing the above-mentioned polypeptide with auxiliary materials.
  • a method for reducing the melanin content in the skin or inhibiting melanin production comprising administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the twelfth aspect of the present invention provides a method for whitening the skin or fading spots, the method comprising administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the thirteenth aspect of the present invention provides a method for treating or preventing pigmentation, which comprises administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • a method for skin antioxidant comprises administering a polypeptide, the above-mentioned antioxidant or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and/or CFFY (SEQ ID NO: 5).
  • the administration methods include but are not limited to oral administration, injection, coating, topical application, etc.
  • One or more amino acids in the amino acid sequence of the polypeptide of the present invention are modified by acetylation, amidation, formylation, hydroxylation, fatty acid chain modification, methylation or phosphorylation.
  • the fatty acid chain is a fatty acid with a carbon chain length of 10-30, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
  • the fatty acid chain is a saturated fatty acid or an unsaturated fatty acid.
  • the fatty acid chain is C 25 H 42 N 2 O 7 (Pal-Glu(OSu)-OH).
  • CPD SEQ ID NO: 2, Cys-Pro-Asp
  • CRVI (SEQ ID NO: 4, Cys-Arg-Val-Ile) has a molecular weight of 489 Da and an isoelectric point of 8.2.
  • CFFY (SEQ ID NO: 5, Cys-Phe-Phe-Tyr) has a molecular weight of 578 Da and an isoelectric point of 5.6.
  • the cosmetics described herein may include lotions, cosmetic milks, facial cleansers, makeup removers, makeup remover oils, makeup remover milks, facial masks, facial creams, essence waters, essence milks, essence oils, essence creams, eye creams, foundations, bath lotions, hair conditioners, hair waters, body perfumes, and the like.
  • Cosmetics refer to industrial chemicals or fine chemical products that are applied to any part of the human body, such as the skin, hair, nails, lips, and teeth, by smearing, spraying, or other similar methods for the purpose of cleansing, maintaining, beautifying, modifying, or altering the appearance of the body, or correcting body odor, to maintain a healthy appearance.
  • the medicines described in the present invention include but are not limited to: freckle-removing medicines, pigmentation-treating medicines or light-blocking agents (for internal or external use).
  • the tissue filler of the present invention can be used for repairing and filling soft tissues of the face, ears, chest, hands, joints, etc.
  • the food of the present invention includes but is not limited to: health care products, oral liquids, and is used for improving pigmentation, resisting oxidative free radicals, whitening, etc.
  • the polypeptide provided by the present invention has extremely strong antioxidant activity, with an oxygen free radical scavenging rate of between 70% and 90%; it has an extremely strong tyrosinase activity inhibitory effect, with a half-inhibitory concentration (IC50) between 7.35 and 10.2 ⁇ mol/L, which is much lower than the half-inhibitory concentration of 902 ⁇ mol/L of the commonly used tyrosinase inhibitor arbutin; at the same time, the polypeptide has an extremely strong ability to inhibit the production of melanin in B16 cells.
  • IC50 half-inhibitory concentration
  • the relative content of melanin is between 52% and 67%; this is better than or equivalent to arbutin, indicating that the polypeptide of the present invention has an excellent ability to inhibit melanin production, can block the formation of melanin in cells from the source, and can effectively reduce melanin and achieve whitening or freckle removal effects.
  • the polypeptide provided by the present invention has low cytotoxicity. Under the conditions of 0.5-2 mg/mL, it does not inhibit the growth of L929 cells and is non-cytotoxic. Even at a high concentration dose (2 mg/mL), the growth of L929 cells is still not inhibited.
  • the polypeptide is highly safe and stable and has no cytotoxicity, which can solve the problem that high concentrations of arbutin are prone to produce adverse reactions.
  • the polypeptide provided by the present invention has a small molecular weight and excellent transdermal performance; after modification with fatty acid side chains, the transdermal effects of polypeptides CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) or CFFY (SEQ ID NO: 5) are increased to 3.0, 4.9 and 3.2 times, respectively, making them particularly suitable for use in whitening and freckle-removing cosmetics.
  • the present invention can efficiently bind to the tyrosinase target, showing excellent tyrosinase activity inhibition effect. At the same time, it has a strong ability to inhibit melanin production, and can also take into account free radical scavenging and antioxidant effects.
  • the preparation process is simple and the cost is low. It can be widely used in the fields of whitening and freckle removal cosmetics, agricultural products, foods, tissue fillers, pharmaceuticals, etc.
  • Figure 2 Cytotoxicity results of different concentrations of PEP-2, PEP-4, PEP-5 and arbutin.
  • Figure 3 The half-maximal effective concentration of PEP-2 for inhibiting melanin production in B16 cells.
  • Figure 4 The half-maximal effective concentration of PEP-4 for inhibiting melanin production in B16 cells.
  • Figure 5 The half-maximal effective concentration of PEP-5 for inhibiting melanin production in B16 cells.
  • Figure 6 Chromatogram of fatty acid side chain modification of PEP-2, where the left figure is the detection diagram of fatty acid side chain modification PEP2C, and the right figure is the detection diagram of PEP2 (left peak) and modified PEP2C (right peak).
  • FIG. 7 Chromatogram of PEP-4 fatty acid side chain modification (PEP4C).
  • FIG. 8 Chromatogram of PEP-5 fatty acid side chain modification (PEP5C).
  • Figure 9 Comparison of transdermal effects of PEP-2 before and after fatty acid side chain modification.
  • Figure 10 Comparison of transdermal effects of PEP-4 before and after fatty acid side chain modification.
  • Figure 11 Comparison of transdermal effects of PEP-5 before and after fatty acid side chain modification.
  • Peptide sequence (PEP sequence)
  • X 2 is the second amino acid of the above polypeptide.
  • the polypeptide was cleaved from the resin using a cleavage solution (1% by volume of trifluoroacetic acid (TFA) in DCM) to obtain a crude polypeptide.
  • a cleavage solution 1% by volume of trifluoroacetic acid (TFA) in DCM
  • the purified polypeptide finished product qualified liquid is freeze-dried using a vacuum freeze dryer to obtain a finished powder.
  • the backbone polypeptide (PEP series) and the fatty acid activation product (Pal-Glu(OSu)-OH, C 25 H 42 N 2 O 7 ) were mixed at a molar ratio of 1:1.5, with a reaction temperature of 25° C., a reaction pH of 4.5, a reaction time of 2 h, and a reaction termination pH of 11.5.
  • the purified polypeptide product liquid is freeze-dried using a vacuum freeze dryer to obtain a finished product powder.
  • L929 cells from ATCC
  • MEM medium PBS
  • HSS Hank's balanced salt solution
  • FBS penicillin-streptomycin
  • FBS fetal bovine serum
  • trypsin containing EDTA
  • DMSO DMSO
  • the experimental groups were selected from arbutin and PEP series peptides (PEP-1 to PEP-15), each of which was added at 2 mg/mL, and a blank control was used.
  • test groups were treated with 100% (2 mg/mL), 75% (1.5 mg/mL), 50% (1 mg/mL), and 25% (0.5 mg/mL) concentrations.
  • negative control use culture medium as a blank. Incubate cells for 24 hours (5% CO 2 , 37°C, >90% humidity).
  • OD 570e average optical density of 100% extract of test sample
  • OD 570b average optical density of blank
  • peptides PEP-2, PEP-4, and PEP-5 did not inhibit the growth of L929 cells, indicating that the above peptides were non-cytotoxic.
  • arbutin in the control group significantly inhibited the growth of L929 cells at a low concentration of 0.5 mg/mL, with a cell survival rate of only 43%, indicating a certain degree of cytotoxicity.
  • the concentration was increased to 2 mg/mL, the cell survival rate was less than 10%, indicating strong cytotoxicity. This also verifies that arbutin can cause adverse reactions such as irritation and skin whitening at high concentrations, limiting its application in whitening products such as cosmetics and foods.
  • a 96-well microtiter plate was set up with solvent background wells ( Ta ), solvent reaction wells ( Tb ), sample background wells ( Tc ), and sample reaction wells ( Td ).
  • Group Ta was the solvent background group, without the addition of the substrate L-tyrosine solution or sample solution;
  • group Tb was the solvent reaction group, with the addition of the substrate L-tyrosine solution but no sample solution;
  • group Tc was the sample background group, without the addition of the substrate L-tyrosine solution but with the addition of the sample solution;
  • group Td was the sample reaction group, with both the substrate L-tyrosine solution and the sample solution.
  • Three replicates were performed for each group; a blank group and a control were also set up.
  • the sample is PEP-1 to PEP-11 polypeptides
  • the solvent is a PBS solution with a pH of about 7.
  • the sample solution is a polypeptide solution formed by dissolving the polypeptide in a solvent.
  • Y is the tyrosinase activity inhibition rate
  • Ad is the absorbance of the sample reaction well
  • Ac is the absorbance of the sample background well
  • Ab is the average absorbance of the solvent reaction well
  • Aa is the average absorbance of the solvent background well.
  • Mouse melanoma cells B16
  • DMEM high glucose
  • PBS Hank's balanced salt solution
  • HBSS Hank's balanced salt solution
  • FBS penicillin-streptomycin
  • FBS fetal bovine serum
  • trypsin containing EDTA
  • DMSO dimethyl sulfoxide
  • MTT thiazolyl blue
  • the PEP series polypeptides were all prepared by the method of Example 1.
  • Well-grown B16 cells were passaged onto 6 mm dishes and plated at a density of 250,000 cells/well in a 4 mL volume per well. After plating, the cells were incubated in a CO2 incubator for 24 hours, and the culture medium in the cell culture wells was discarded.
  • the peptide samples were prepared using DMEM complete medium and cultured at 37°C, 5% CO2 .
  • the blank group the peptide samples were prepared using DMEM complete medium and cultured at 37°C, 5% CO2 .
  • the melanin inhibition ability is the strongest among the polypeptides, while the melanin content of the commonly used arbutin is 65%; that is, at the same concentration, the melanin content of polypeptides PEP-2 and PEP-4 is significantly lower than that of arbutin, which also shows that the polypeptides PEP-2 and PEP-4 of the present invention have excellent ability to inhibit melanin production, can block the formation of melanin in cells from the source, and have significant effects of reducing melanin and whitening and removing spots.
  • polypeptide PEP-5 is basically equivalent to that of arbutin, but based on arbutin, it will produce cytotoxicity at a concentration of 0.5 mg/mL (as shown in Test Example 1), while the polypeptides of the present invention are non-cytotoxic, and even at high concentrations (2 mg/mL), they still do not produce obvious cytotoxicity. Therefore, the polypeptide PEP-5 has the advantages of safety, stability, and good biocompatibility, and can replace arbutin and be used in whitening food, medicine or cosmetics.
  • PBS antioxidant Trolox
  • ABTS method total antioxidant capacity detection kit
  • the polypeptide sample to be tested was prepared using PBS so that the concentration of the sample to be tested was 1 mmol/L.
  • Free radical scavenging ability sample measured value / 1mmol/L Trolox measured value ⁇ 100%
  • the results are shown in Table 6.
  • the free radical scavenging ability of PEP-2, PEP-4, PEP-5, PEP-7, and PEP-9 is about 85% or more.
  • PEP-2, PEP-4, and PEP-5 have extremely strong antioxidant properties and also have a strong ability to inhibit melanin production (Test Example 3).
  • the sample is added to the pig skin, and the transdermal effect is detected using a transdermal diffusion test system.
  • transdermal effects of the fatty chain modified polypeptides PEP-2C, PEP-4C, and PEP-5C were 3.0, 4.9, and 3.2 times that of the unmodified fatty acid chain PEP-2, PEP-4, and PEP-5 (Example 1), respectively. This shows that the polypeptide has a good transdermal effect and is particularly suitable for use in the field of whitening cosmetics.
  • the peptides PEP-2, PEP-4, and PEP-5 were screened for their ability to stably and effectively inhibit tyrosinase, inhibit melanin production, and scavenge free radicals. Further, the effects of variations in the amino acid sequence within the peptides on their performance were investigated.
  • the peptide sample preparation method was the same as in Example 1; the methods for evaluating tyrosinase inhibition, melanin production, and free radical scavenging effects are described in Test Examples 2-4.
  • the performance test results for the different peptide samples are shown in Table 7.
  • Test Example 7 Effect of (PEP-4) Peptide Sequence Order
  • Test Example 8 Effect of (PEP-5) Peptide Sequence Order
  • the peptide preparation and testing methods were as described above.
  • the amino acid sequence of the peptide PEP-5 was adjusted.
  • the performance test results of different peptide samples are shown in Table 9.
  • polypeptides PEP-2, PEP-4, and PEP-5 of the present invention can simultaneously meet the performance requirements of whitening and antioxidant properties, have excellent melanin inhibition and free radical scavenging capabilities, and can meet the use requirements of whitening and antioxidant products.

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Abstract

L'invention concerne un groupe de polypeptides, qui peut être utilisé en tant qu'inhibiteur de la tyrosinase. Les polypeptides ont pour effet d'inhiber l'activité de la tyrosinase, de réduire la sécrétion de mélanine, de résister à l'oxydation, de blanchir la peau, d'éliminer les taches, etc. Les polypeptides peuvent être largement utilisés dans des médicaments antioxydants et dans la préparation de produits cosmétiques de blanchiment de la peau pour résister à la mélanogenèse et à des taches d'éclaircissement en tant que nouvelle matière première, et présente de bonnes perspectives d'application.
PCT/CN2025/081717 2024-03-15 2025-03-11 Polypeptides et leur utilisation Pending WO2025190245A1 (fr)

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CN202410299714.0A CN120647713A (zh) 2024-03-15 2024-03-15 多肽及其应用

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