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WO2024000088A1 - Polypeptide éclaircissant dérivé de perles et son utilisation - Google Patents

Polypeptide éclaircissant dérivé de perles et son utilisation Download PDF

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Publication number
WO2024000088A1
WO2024000088A1 PCT/CN2022/101398 CN2022101398W WO2024000088A1 WO 2024000088 A1 WO2024000088 A1 WO 2024000088A1 CN 2022101398 W CN2022101398 W CN 2022101398W WO 2024000088 A1 WO2024000088 A1 WO 2024000088A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
nevus
whitening
pearl
seq
Prior art date
Application number
PCT/CN2022/101398
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English (en)
Chinese (zh)
Inventor
叶成龙
王菁
杨安全
张雅琪
郭路遥
费程霞
胡秋艺
莫家欢
Original Assignee
欧诗漫生物股份有限公司
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.)
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Publication date
Application filed by 欧诗漫生物股份有限公司 filed Critical 欧诗漫生物股份有限公司
Priority to PCT/CN2022/101398 priority Critical patent/WO2024000088A1/fr
Publication of WO2024000088A1 publication Critical patent/WO2024000088A1/fr

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    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • 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 invention belongs to the field of biomedicine and relates to a pearl-derived whitening polypeptide and its application.
  • Tyrosinase (EC 1.14.18.1) is a copper enzyme and a key enzyme in the synthesis of melanin. It catalyzes the hydroxylation of monophenols to o-diphenols and then the oxidation of o-diphenols to o-quinones, ultimately leading to the formation of melanin.
  • Some whitening ingredients achieve skin whitening effects by inhibiting tyrosinase, such as ascorbic acid, kojic acid, hydroquinone and arbutin, but they also have certain side effects.
  • Ascorbic acid is widely used as a whitening agent, but it loses its efficacy after a rapid redox reaction.
  • Kojic acid, hydroquinone, and arbutin are widely used in cosmetics to achieve skin whitening effects, however, previous studies have reported genotoxic and carcinogenic effects of these compounds. Therefore, it is of great significance to find natural, safe and efficient whitening agents.
  • Pearl is a precious organic gemstone whose main component is CaCO 3 with a small amount of organic matter and water. At present, there are many documents proving that pearls have whitening effect.
  • the present invention provides a whitening polypeptide, which is selected from one or more polypeptides with amino acid sequences such as SEQ ID NO.1-5; the amino terminal sequence of SEQ ID NO.1 is EGGGGFG ; The amino terminal sequence of SEQ ID NO.2 is MHFHEVN; the amino terminal sequence of SEQ ID NO.3 is GGFDGGF; the amino terminal sequence of SEQ ID NO.4 is ASAGGFG; the amino terminal sequence of SEQ ID NO.5 is GSEHRIN.
  • the invention provides a pearl-derived whitening polypeptide, whose amino acid sequence is shown as SEQ ID NO: 1-SEQ ID NO: 5.
  • the molecular docking results show that the polypeptide SEQ ID NO: 1-SEQ ID NO: 5 and Tyrosinase (PDB: 2Y9X) has a strong affinity, indicating that the pearl-derived whitening peptide has potential tyrosinase inhibitory activity.
  • the present invention provides a method for preparing the aforementioned whitening polypeptide, which method includes using a polypeptide synthesizer to synthesize the target polypeptide according to the amino acid sequence; in some specific embodiments, using a polypeptide solid-phase synthesis reactor to synthesize the target polypeptide.
  • the present invention uses the aforementioned whitening polypeptides to prepare cosmetics or pharmaceuticals with melanin inhibitory activity; in some embodiments, the aforementioned polypeptides are added to the cell culture medium, and the cells after the polypeptide treatment are measured.
  • the melanin content of B16 melanoma cells showed that the polypeptides showed significant ability to inhibit melanin production, indicating that the polypeptides SEQ ID NO. 1-5 provided by the invention have potential whitening effects.
  • Existing polypeptides on the market have all undergone melanin-promoting pretreatment with B16 cells in the melanin-inhibiting activity test. After being tested using the method of this embodiment, they have almost no melanin-inhibiting activity; in the embodiment of the present invention, the B16 provided
  • the cells are cells that are in a natural state and have not been subjected to melanin-promoting tests. They can better reflect the true melanin inhibitory activity of the active peptide composition and more truly reflect the melanin inhibitory activity of the highly active polypeptide mixture.
  • the cosmetics or pharmaceuticals include cosmetics or pharmaceuticals that have the effect of whitening or treating or preventing melanin pigmentation diseases; specifically, the hypermelanin pigmentation diseases include but are not limited to chloasma, freckles, and age spots. , spots, café-au-lait spots, Baker’s nevus, nevus, Mongolian spot, nevus of Ohta, acquired bilateral nevus of Ohta, Ito nevus, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus Moles, Spitz nevus, dysplastic nevus, sun spots.
  • the hypermelanin pigmentation diseases include but are not limited to chloasma, freckles, and age spots. , spots, café-au-lait spots, Baker’s nevus, nevus, Mongolian spot, nevus of Ohta, acquired bilateral nevus of Ohta, It
  • the present invention provides a method for preparing a whitening polypeptide.
  • the whitening polypeptide is one of the polypeptides with an amino acid sequence as shown in SEQ ID NO. 1-5.
  • the preparation method includes using polypeptide synthesis according to the amino acid sequence. instrument synthesis.
  • a polypeptide mixture is obtained by extracting pearls, and molecular docking is performed through Discovery Sdudio 2019 software to screen out pearl polypeptides with potential affinity for tyrosinase (PDB: 2Y9X).
  • PDB tyrosinase
  • the inventors took the polypeptide with strong affinity to tyrosinase in the molecular docking results as the target polypeptide, and chemically synthesized it through solid-phase synthesis. Finally, the synthesized peptide was added to the cell culture medium, the melanin content of B16 melanoma cells was measured, and highly active whitening peptides SEQ ID NO. 1-5 were screened out.
  • the polypeptide provided by the invention is a pearl polypeptide.
  • FIG. 1 3D structure diagram of tyrosinase (PDB: 2Y9X).
  • Figure 2 shows the melanin content of B16 melanoma cells after treatment with pearl-derived whitening peptide SEQ ID NO.1-5.
  • Table 1 is a comparison table of amino acid name definitions cited in the present invention.
  • Preprocessing of target protein Download the PDB format file of tyrosinase protein (PDB: 2Y9X) in the PDB database (https://www.pdbus.org/), then open the file with Discovery Sdudio 2019 software, and click on " Click "Prepare Protein” in Macromolecules” to perform protein preprocessing.
  • the new protein obtained after preprocessing has a total of 66 sites.
  • the polypeptide has a higher possibility of docking with sites 1-10 of the tyrosinase protein. Therefore, sites 1-10 of the tyrosinase were selected as docking sites, respectively with the polypeptide.
  • Perform rigid docking (LibDock), and record the number of Pose docked at each site and the highest rigid docking score (LibDockScore). The results are shown in Table 1.
  • the pearl-derived whitening peptide SEQ ID NO.1-5 can be successfully docked with multiple sites of tyrosinase, and has a high pose number, especially EGGGGFG and ASAGGFG, positions 1-10 The cumulative number of poses reached 751 and 835 respectively.
  • MHFHEVN, GGFDGGF and GSEHRIN have higher docking scores, with the highest values being 230.206, 216.46 and 224.105 respectively.
  • the above results show that the pearl polypeptide has a strong affinity with tyrosinase, suggesting that it has potential whitening effect.
  • Step 1 Weigh 0.3mmol MBHA amino resin into the reaction tube, swell the resin with dichloromethane (DCM) for 40 minutes, clean the resin with N,N-dimethylformamide (DMF) and drain it, and clean it 5 times in total.
  • DCM dichloromethane
  • DMF N,N-dimethylformamide
  • Step 2 Use 20% piperidine (dissolved in DMF) to remove the Fmoc protecting group for 3 minutes, drain it, wash with DMF and drain it, use 20% piperidine again to remove the Fmoc protecting group for 7 minutes, drain it, and clean the resin with DMF Then drain it and wash it 5 times in total.
  • Step 3 Add 0.9mmol Fmoc-Asn-OH, 0.85mmol HBTU, 0.85mmol HOBT and 1.8mmol Dipea to the reaction tube, react at room temperature for 45 minutes, drain, wash the resin with DMF and drain, wash 5 times in total.
  • Step 4 Put a small amount of resin into a test tube, wash it twice with DMF and MeOH, add about 0.3 mL of 2% ninhydrin indicator, and observe the color change of the resin after heating for 5 minutes. If no blue color appears, the reaction is complete. Blue indicates that the reaction is incomplete and needs to continue.
  • Step 5 Replace Fmoc-Asn-OH in step 3 with Fmoc-Val-OH, Fmoc-Glu-OH, Fmoc-His-OH, Fmoc-Phe-OH, Fmoc-His-OH, Fmoc-Met- OH, and repeat steps 2-4 until the amino acids on the polypeptide sequence are completely connected.
  • Step 6 After deprotecting the last connected amino acid Fmoc-Met-OH, wash the resin 5 times with DMF and then 5 times with DCM.
  • Step 7 Add 5 mL of TFA:H 2 O:TIPS (95:2.5:2.5) to the reaction tube, react at room temperature for 2 hours, filter, rinse with a small amount of TFA and collect the liquid into a centrifuge tube. Slowly add diethyl ether dropwise to the liquid. During this period, a light yellow solid will be generated. Place the centrifuge tube in a -20°C refrigerator for 2 hours, centrifuge and discard the supernatant. Add diethyl ether again, centrifuge and discard the supernatant.
  • Step 8 Dry the obtained solid in a fume hood, then dissolve it in water and pass it through a 0.22 ⁇ m PES filter membrane, and purify it with SemiPrep RP-HPLC.
  • the mobile phase is water containing 0.1% TFA (Buffer A) and water containing 0.1% TFA.
  • TFA's acetonitrile (Buffer B) the detection wavelength is set to 214nm.
  • the collected peptides were characterized and confirmed using Analytical RP-HPLC and MALDI-TOF-MS, and then freeze-dried with a freeze dryer to obtain the target peptide SEQ ID NO. 2 MHFHEVN.
  • SEQ ID NO.1 EGGGGFG
  • SEQ ID NO.3 GGFDGGF
  • SEQ ID NO.4 ASAGGFG
  • SEQ ID NO.5: GSEHRIN is also synthesized using a similar method.
  • B16 melanoma cells grown in DMEM complete medium and in the logarithmic growth phase were seeded into 100mm cell culture dishes at a density of (10-15) ⁇ 10 4 cells/mL, and 10 mL of complete culture medium was added to each culture dish. , placed in a carbon dioxide incubator at 37°C and 5% CO 2 concentration. After 24 hours, add polypeptide solution (dissolved in PBS) to the culture dish. The final concentration of the polypeptide is 100mg/L (100ppm). Add an equal amount of PBS solution to the control group, and then return it to the incubator to continue culturing. After 24 hours, the cells were digested with trypsin and centrifuged to collect the cells.
  • pearl-derived whitening peptide SEQ ID NO.1-5 can significantly reduce melanin content (P ⁇ 0.01), reducing it by 27.03%, 32.43%, and 31.23% respectively. , 26.72% and 22.02%, indicating that the polypeptides have strong whitening effects.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un polypeptide éclaircissant dérivé de perles et son utilisation. Le polypeptide est un polypeptide ayant une séquence d'acides aminés selon l'une quelconque des SEQ ID NO 1 à 5. Le résultat d'amarrage moléculaire montre que le polypeptide a une affinité relativement forte avec la tyrosinase, indiquant que le polypeptide a une capacité potentielle d'inhibition de la tyrosinase, de façon à inhiber la génération de mélanine. Des expériences cellulaires montrent que le polypeptide peut inhiber de manière remarquable la génération de mélanine de cellules de mélanome B16, de telle sorte que le polypeptide dérivé de perle selon la présente invention a un effet éclaircissant potentiel. De plus, le polypeptide dérivé de perles est un peptide actif naturel, présente les avantages d'une bonne stabilité, d'une faible cytotoxicité et d'être facile à absorber par le corps humain, etc, et présente de grandes perspectives d'application dans la préparation d'un produit de soin de la peau ayant un effet d'éclaircissement.
PCT/CN2022/101398 2022-06-27 2022-06-27 Polypeptide éclaircissant dérivé de perles et son utilisation WO2024000088A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117801070A (zh) * 2024-03-01 2024-04-02 中国农业大学 一种美白祛斑消水肿青稞酒糟肽及其制备方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010112713A2 (fr) * 2009-04-02 2010-10-07 Isp Investments Inc. Nouveaux peptides eclaircissants activateurs du proteasome et compositions les contenant
US20170246098A1 (en) * 2014-09-01 2017-08-31 Institut Curie Skin whitening peptide agents
WO2019231175A1 (fr) * 2018-05-28 2019-12-05 주식회사 루비크라운 Peptide à terminaison carboxy amidée ayant une activité inhibitrice de la mélanogenèse et composition le comprenant
CN111741968A (zh) * 2019-01-25 2020-10-02 Neomics 株式会社 具有皮肤增白活性的新型多肽及其用途
CN112689641A (zh) * 2019-08-20 2021-04-20 凯尔格恩有限公司 具有皮肤美白活性的肽以及其用途
WO2022038555A1 (fr) * 2020-08-20 2022-02-24 Colosseum Combinatorial Chemistry Centre For Technology Societa' A Responsabilita' Limitata In Forma Abbreviata C4T S.R.L. Peptide pour applications thérapeutiques dans le domaine dermatologique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010112713A2 (fr) * 2009-04-02 2010-10-07 Isp Investments Inc. Nouveaux peptides eclaircissants activateurs du proteasome et compositions les contenant
US20170246098A1 (en) * 2014-09-01 2017-08-31 Institut Curie Skin whitening peptide agents
WO2019231175A1 (fr) * 2018-05-28 2019-12-05 주식회사 루비크라운 Peptide à terminaison carboxy amidée ayant une activité inhibitrice de la mélanogenèse et composition le comprenant
CN111741968A (zh) * 2019-01-25 2020-10-02 Neomics 株式会社 具有皮肤增白活性的新型多肽及其用途
CN112689641A (zh) * 2019-08-20 2021-04-20 凯尔格恩有限公司 具有皮肤美白活性的肽以及其用途
WO2022038555A1 (fr) * 2020-08-20 2022-02-24 Colosseum Combinatorial Chemistry Centre For Technology Societa' A Responsabilita' Limitata In Forma Abbreviata C4T S.R.L. Peptide pour applications thérapeutiques dans le domaine dermatologique

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117801070A (zh) * 2024-03-01 2024-04-02 中国农业大学 一种美白祛斑消水肿青稞酒糟肽及其制备方法和应用
CN117801070B (zh) * 2024-03-01 2024-06-07 中国农业大学 一种美白祛斑消水肿青稞酒糟肽及其制备方法和应用

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