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WO2024000088A1 - 一种珍珠来源美白多肽及其应用 - Google Patents

一种珍珠来源美白多肽及其应用 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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polypeptide
nevus
whitening
pearl
seq
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French (fr)
Inventor
叶成龙
王菁
杨安全
张雅琪
郭路遥
费程霞
胡秋艺
莫家欢
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Osmun Biology Co Ltd
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Osmun Biology Co Ltd
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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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Abstract

本发明公开了一种珍珠来源美白多肽及其应用，所述多肽是具有SEQ ID NO.1-5中任一项所述的氨基酸序列的多肽。分子对接结果发现，所述多肽与酪氨酸酶均有较强的亲和力，表明其具有潜在的酪氨酸酶抑制能力，从而抑制黑色素生成；通过细胞实验进一步验证，所述多肽均能显著抑制B16黑色素瘤细胞的黑色素生成，表明本发明的珍珠来源多肽具有潜在的美白功效；同时，珍珠多肽作为天然活性肽，具有稳定性好，细胞毒性弱、易被人体吸收等优点，在制备具有美白功效的护肤品中具有较大的应用前景。

Description

一种珍珠来源美白多肽及其应用 技术领域
本发明属于生物医药领域，涉及一种珍珠来源美白多肽及其应用。
背景技术
随着生活水平的日益提高，人们更加注重个人形象和对皮肤的保养。美白是现代生活中的一个热门话题，许多人会投入大量的时间和精力，但是效果往往不尽人意。酪氨酸酶(EC 1.14.18.1)是一种铜酶，是黑色素合成过程中的关键酶。它催化单酚羟基化为邻二酚，然后将邻二酚氧化为邻醌，最终导致黑色素的生成。
一些美白成分通过抑制酪氨酸酶从而达到皮肤美白效果，例如抗坏血酸、曲酸、对苯二酚和熊果苷等，但它们也存在一定的副作用。抗坏血酸作为增白剂被广泛应用，但其在快速氧化还原反应后会失去功效。曲酸、对苯二酚和熊果苷被广泛应用于化妆品中以实现皮肤美白效果，然而，之前的研究报告了这些化合物的基因毒性和致癌作用。因此，寻找天然、安全、高效的美白剂具有重要意义。珍珠是一种珍贵的有机宝石，其主要成分为CaCO 3，另有少量有机质和水。目前，已有不少文献证明珍珠具有美白功效，同时我们前期通过实验证明珍珠提取物可以抑制酪氨酸酶的活性，从而减少黑色素的生成。据报道，天然来源的蛋白质衍生物、多肽和蛋白质水解物具有美白效果，并应用于治疗皮肤病。因此，从珍珠提取物中分离得到的多肽可能具有潜在的美白功效。我们通过质谱鉴定出1437种珍珠多肽，确定了这些多肽的氨基酸序列之后，利用分子对接技术预测出与酪氨酸酶具有潜在亲和活性的多肽，并通过固相合成法进行合成，通过体外细胞实验筛选出具有黑色素抑制活性的多肽，从而大幅开阔了珍珠在美白中的应用前景。
发明内容
第一方面，本发明提供一种美白多肽，所述多肽选自氨基酸序列如SEQ ID NO.1-5所示的多肽中的一种或多种；SEQ ID NO.1的氨基端序列为EGGGGFG；SEQ ID NO.2的氨基端序列为MHFHEVN；SEQ ID NO.3的氨基端序列为GGFDGGF；SEQ ID NO.4的氨基端序列为ASAGGFG；SEQ ID NO.5的氨基端序列为GSEHRIN。
以所述的珍珠来源美白多肽为核心，任何对其进行的相应的调整或修饰都属于本发明的保护范围。
本发明提供的一种珍珠来源美白多肽，其氨基酸序列如SEQ ID NO：1-SEQ ID NO：5所示，通过分子对接结果表明，所述多肽SEQ ID NO：1-SEQ ID NO：5与酪氨酸酶(PDB： 2Y9X)具有较强的亲和力，表明珍珠来源美白多肽具有潜在的酪氨酸酶抑制活性。
第二方面，本发明提供前述所述美白多肽的制备方法，该制备方法包括根据氨基酸序列，使用多肽合成仪合成；在一些具体实施例中，利用多肽固相合成反应器合成目标多肽。
第三方面，本发明将前述所述的美白多肽用于制备具有黑色素抑制活性的化妆品或药品；在一些实施例中，将前述所述的多肽添加到细胞培养基中，测定多肽处理后细胞的B16黑色素瘤细胞的黑色素含量，结果表明所述多肽均表现出显著的抑制黑色素生成能力，表明了所述本发明提供的多肽SEQ ID NO.1-5具有潜在的美白功效。
市场上现有的多肽在抑制黑色素活性试验中均进行了B16细胞促黑预处理，将其利用本实施例方式进行试验后，几乎没有黑色素抑制活性；在本发明的实施例中，提供的B16细胞为处于自然状态未进行促黑试验的细胞，其更能体现活性肽组合物真实的黑色素抑制活性，更加真实反映高活性多肽混合物的黑色素抑制活性。
在一些具体实施例中，所述的化妆品或药品包括具有美白或治疗或预防黑色素沉着疾病功效的化妆品或药品；具体地，所述黑色素过度沉着疾病包括但不限于黄褐斑、雀斑、老人斑、斑点、牛奶咖啡斑、贝克痣、斑痣、蒙古斑、太田痣、获得性双侧太田痣样斑、伊藤痣、蓝痣、交界痣、混合痣、皮内痣、晕痣、先天性黑色素细胞痣、斯皮茨痣、发育不良性痣、晒斑。
第四方面，本发明提供一种美白多肽的制备方法，该美白多肽为氨基酸序列如SEQ ID NO.1-5所示的多肽中的一种，所述制备方法包括根据氨基酸序列，使用多肽合成仪合成。
在本发明一些具体实施例中，通过对珍珠进行提取获得多肽混合物，通过Discovery Sdudio 2019软件进行分子对接，筛选出与酪氨酸酶(PDB：2Y9X)具有潜在亲和力的珍珠多肽。在此基础上，本发明人将分子对接结果中与酪氨酸酶具有较强亲和力的多肽作为目标多肽，并通过固相合成法进行化学合成。最终将合成得到的多肽添加到细胞培养基中，测定B16黑色素瘤细胞的黑色素含量，并筛选出具有高活性的美白多肽SEQ ID NO.1-5。
本发明提供的多肽为珍珠多肽，其作为天然活性肽，具有稳定性好，细胞毒性弱、易被人体吸收等优点，在制备具有美白功效的护肤品中具有较大的应用前景。
附图说明
图1酪氨酸酶(PDB：2Y9X)的3D结构图。
图2为B16黑色素瘤细胞经珍珠来源美白多肽SEQ ID NO.1-5处理后的黑色素含量。
具体实施方式
下面结合说明书附图及具体实施例子对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。
表1本发明所引用氨基酸名称定义对照表。
谷氨酸 E(Glu)
甘氨酸 G(Gly)
苯丙氨酸 F(Phe)
蛋氨酸 M(Met)
组氨酸 H(His)
缬氨酸 V(Val)
天冬酰胺 N(Asn)
天冬酰胺 D(Asp)
丙氨酸 A(Ala)
丝氨酸 S(Ser)
精氨酸 R(Arg)
异亮氨酸 I(Ile)
珍珠来源美白多肽EGGGGFG、MHFHEVN、GGFDGGF、ASAGGFG和GSEHRIN通过固相合成法得到，纯度大于95％。
实施例1分子对接
靶蛋白的预处理：在PDB数据库(https://www.pdbus.org/)中下载酪氨酸酶蛋白(PDB:2Y9X)的PDB格式文件，然后用Discovery Sdudio 2019软件打开该文件，在“Macromolecules”中点击“Prepare Protein”，对其进行蛋白预处理，预处理后得到的新蛋白共有66个位点。
多肽配体的准备：利用Chemdraw 19.0软件画出多肽的分子结构，保存为“sdf”格式，然后用Discovery Sdudio 2019软件打开该多肽文件，在“Small Molecules”中点击“Prepare Ligands”，对其进行预处理。
分子对接：根据经验，多肽与酪氨酸酶蛋白的1-10号位点有较高的对接可能性，因此，选择酪氨酸酶的1-10号位点作为对接位点，分别与多肽进行刚性对接(LibDock)，记录每个位点对接的Pose数及最高刚性对接得分(LibDockScore)，结果如表一所示。
表1珍珠来源美白多肽SEQ ID NO.1-5与酪氨酸酶的分子对接结果
Figure PCTCN2022101398-appb-000001
Figure PCTCN2022101398-appb-000002
由表1可见，珍珠来源美白多肽SEQ ID NO.1-5与酪氨酸酶的多个位点均能够成功对接，且具有较高的pose数，尤其是EGGGGFG和ASAGGFG，1-10号位点的累计pose数分别达到了751和835。另外，MHFHEVN、GGFDGGF和GSEHRIN具有较高的对接分值，最高值分别为230.206、216.46和224.105。以上结果表明，所述珍珠多肽与酪氨酸酶之间具有较强的亲和力，提示其具有潜在的美白功效。
实施例2多肽的固相合成(以SEQ ID NO.2MHFHEVN为例)
步骤1：称取0.3mmol MBHA氨基树脂于反应管中，用二氯甲烷(DCM)溶胀树脂40min，用N,N-二甲基甲酰胺(DMF)清洗树脂并抽干，共清洗5次。
步骤2：用20％的哌啶(溶于DMF)脱Fmoc保护基3min，抽干，用DMF清洗并抽干，再次用20％的哌啶脱Fmoc保护基7min，抽干，用DMF清洗树脂并抽干，共清洗5次。
步骤3：向反应管中加入0.9mmol Fmoc-Asn-OH、0.85mmol HBTU、0.85mmol HOBT和1.8mmol Dipea，室温下反应45min，抽干，用DMF清洗树脂并抽干，共清洗5次。
步骤4：取少量树脂于试管中，DMF、MeOH各洗2次，加入约0.3mL的2％茚三酮指示剂，加热5分钟后观察树脂颜色变化，不显蓝色则表明反应完全，显蓝色则表明反应不完全，需要继续反应。
步骤5：将步骤3中的Fmoc-Asn-OH依次替换为Fmoc-Val-OH、Fmoc-Glu-OH、 Fmoc-His-OH、Fmoc-Phe-OH、Fmoc-His-OH、Fmoc-Met-OH，并重复步骤2-4的操作，直至将多肽序列上的氨基酸连接完全。
步骤6：对最后一个连接上的氨基酸Fmoc-Met-OH脱Fmoc保护后，先用DMF清洗树脂5次，再用DCM清洗5次。
步骤7：向反应管中加入5mL的TFA：H 2O：TIPS(95：2.5：2.5)，室温下反应2h，过滤，用少量TFA润洗并将液体收集至离心管中。向液体中缓慢滴加乙醚，期间会生成淡黄色固体，将离心管置于-20℃冰箱中2h，离心弃上清，再次加入乙醚，离心弃上清。
步骤8：将得到的固体置于通风橱中干燥，然后用水溶解并过0.22μm PES滤膜，用SemiPrep RP-HPLC进行纯化，流动相为含有0.1％TFA的水(Buffer A)和含有0.1％TFA的乙腈(Buffer B)，检测波长设为214nm。收集到的多肽用Analytical RP-HPLC和MALDI-TOF-MS进行表征确认后用冻干机冻干，即得到目标多肽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也采用类似的方法合成。
实施例3黑色素抑制实验
将生长于DMEM完全培养基、处于对数生长期的B16黑色素瘤细胞，以(10-15)×10 4个/mL的密度接种于100mm细胞培养皿中，每个培养皿加入10mL完全培养基，置于37℃、5％CO 2浓度的二氧化碳培养箱中培养。24h后向培养皿加入多肽溶液(溶于PBS)，多肽的终浓度为100mg/L(100ppm)，对照组加等量的PBS溶液，然后放回培养箱继续培养。24h后，用胰酶消化并离心收集细胞，弃去上清液，用PBS洗涤2次，加入300μL含有10％DMSO的1M NaOH，置于80℃水浴锅中裂解细胞。30min后，取100μL细胞裂解液至96孔板中，用酶标仪读取405nm处的吸光度，最后将吸光度换算成黑色素含量。
如图2所示，在100mg/L浓度下，珍珠来源美白多肽SEQ ID NO.1-5均能极显著性地降低黑色素含量(P＜0.01)，分别降低了27.03％、32.43％、31.23％、26.72％和22.02％，表明所述多肽均具有较强的美白功效。
Figure PCTCN2022101398-appb-000003
Figure PCTCN2022101398-appb-000004

Claims (7)

  1. 一种美白多肽，其特征在于，所述多肽选自氨基酸序列如SEQ ID NO.1-5所示的多肽中的一种或多种。
  2. 如权利要求1的美白多肽的制备方法，其特征在于，该制备方法包括根据氨基酸序列，使用多肽合成仪合成。
  3. 如权利要求1的美白多肽的制备方法，其特征在于，利用多肽固相合成反应器合成目标多肽。
  4. 如权利要求1所述的美白多肽在制备具有黑色素抑制活性的化妆品或药品中的应用。
  5. 如权利要求1所述的美白多肽在制备具有美白或治疗或预防黑色素沉着疾病功效的化妆品或药品中的应用。
  6. 如权利要求4所述应用，其特征在于，所述黑色素过度沉着疾病选自黄褐斑、雀斑、老人斑、斑点、牛奶咖啡斑、贝克痣、斑痣、蒙古斑、太田痣、获得性双侧太田痣样斑、伊藤痣、蓝痣、交界痣、混合痣、皮内痣、晕痣、先天性黑色素细胞痣、斯皮茨痣、发育不良性痣、晒斑。
  7. 一种美白多肽的制备方法，其特征在于，该美白多肽为氨基酸序列如SEQ ID NO.1-5所示的多肽中的一种，所述制备方法包括根据氨基酸序列，使用多肽合成仪合成。
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